Phenyl substituted thiophenes as estrogenic agents

ABSTRACT

This invention provides estrogen receptor modulators of formula I, having the structure  
                 
 
     wherein,  
     R 1 , R 2 , R 3 , X, and Y are as defined in the specification, or a pharmaceutically acceptable salt thereof.

[0001] This application claims priority from copending provisionalapplication Serial No. 60/348,853, filed Jan. 15, 2002, the entiredisclosure of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to phenyl substituted thiphenes, which areuseful as estrogenic agents.

[0003] The pleiotropic effects of estrogens in mammalian tissues havebeen well documented, and it is now appreciated that estrogens affectmany organ systems [Mendelsohn and Karas, New England Journal ofMedicine 340: 1801-1811 (1999), Epperson, et al., Psychosomatic Medicine61: 676-697 (1999), Crandall, Journal of Womens Health & Gender BasedMedicine 8: 1155-1166 (1999), Monk and Brodaty, Dementia & GeriatricCognitive Disorders 11: 1-10 (2000), Hum and Macrae, Journal of CerebralBlood Flow & Metabolism 20: 631-652 (2000), Calvin, Maturitas 34:195-210 (2000), Finking, et al., Zeitschrift fur Kardiologie 89: 442-453(2000), Brincat, Maturitas 35: 107-117 (2000), Al-Azzawi, PostgraduateMedical Journal 77: 292-304 (2001)]. Estrogens can exert effects ontissues in several ways, and the most well characterized mechanism ofaction is their interaction with estrogen receptors leading toalterations in gene transcription. Estrogen receptors areligand-activated transcription factors and belong to the nuclear hormonereceptor superfamily. Other members of this family include theprogesterone, androgen, glucocorticoid and mineralocorticoid receptors.Upon binding ligand, these receptors dimerize and can activate genetranscription either by directly binding to specific sequences on DNA(known as response elements) or by interacting with other transcriptionfactors (such as AP1), which in turn bind directly to specific DNAsequences [Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall,et al., Journal of Biological Chemistry 276: 36869-36872 (2001),McDonnell, Principles Of Molecular Regulation. p351-361(2000)]. A classof “coregulatory” proteins can also interact with the ligand-boundreceptor and further modulate its transcriptional activity [McKenna, etal., Endocrine Reviews 20: 321-344 (1999)]. It has also been shown thatestrogen receptors can suppress NFκB-mediated transcription in both aligand-dependent and independent manner [Quaedackers, et al.,Endocrinology 142: 1156-1166 (2001), Bhat, et al., Journal of SteroidBiochemistry & Molecular Biology 67: 233-240 (1998), Pelzer, et al.,Biochemical & Biophysical Research Communications 286: 1153-7 (2001)].

[0004] Estrogen receptors can also be activated by phosphorylation. Thisphosphorylation is mediated by growth factors such as EGF and causeschanges in gene transcription in the absence of ligand [Moggs andOrphanides, EMBO Reports 2: 775-781 (2001), Hall, et al., Journal ofBiological Chemistry 276: 36869-36872 (2001)].

[0005] A less well-characterized means by which estrogens can affectcells is through a so-called membrane receptor. The existence of such areceptor is controversial, but it has been well documented thatestrogens can elicit very rapid non-genomic responses from cells. Themolecular entity responsible for transducing these effects has not beendefinitively isolated, but there is evidence to suggest it is at leastrelated to the nuclear forms of the estrogen receptors [Levin, Journalof Applied Physiology 91: 1860-1867 (2001), Levin, Trends inEndocrinology & Metabolism 10: 374-377 (1999)].

[0006] Two estrogen receptors have been discovered to date. The firstestrogen receptor was cloned about 15 years ago and is now referred toas ERα [Green, et al., Nature 320: 134-9 (1986)]. The second form of theestrogen receptor was found comparatively recently and is called ERβ[Kuiper, et al., Proceedings of the National Academy of Sciences of theUnited States of America 93: 5925-5930 (1996)]. Early work on ERβfocused on defining its affinity for a variety of ligands and indeed,some differences with ERβ were seen. The tissue distribution of ERβ hasbeen well mapped in the rodent and it is not coincident with ERα.Tissues such as the mouse and rat uterus express predominantly ERα,whereas the mouse and rat lung express predominantly ERβ [Couse, et al.,Endocrinology 138: 4613-4621 (1997), Kuiper, et al., Endocrinology 138:863-870 (1997)]. Even within the same organ, the distribution of ERα andERβ can be compartmentalized. For example, in the mouse ovary, ERβ ishighly expressed in the granulosa cells and ERα is restricted to thethecal and stromal cells [Sar and Welsch, Endocrinology 140: 963-971(1999), Fitzpatrick, et al., Endocrinology 140: 2581-2591 (1999)].However, there are examples where the receptors are coexpressed andthere is evidence from in vitro studies that ERα and ERβ can formheterodimers [Cowley, et al., Journal of Biological Chemistry 272:19858-19862 (1997)].

[0007] A large number of compounds have been described that either mimicor block the activity of 17β-estradiol. Compounds having roughly thesame biological effects as 17β-estradiol, the most potent endogenousestrogen, are referred to as “estrogen receptor agonists”. Those which,when given in combination with 17β-estradiol, block its effects arecalled “estrogen receptor antagonists”. In reality there is a continuumbetween estrogen receptor agonist and estrogen receptor antagonistactivity and indeed some compounds behave as estrogen receptor agonistsin some tissues and estrogen receptor antagonists in others. Thesecompounds with mixed activity are called selective estrogen receptormodulators (SERMS) and are therapeutically useful agents (e.g. EVISTA)[McDonnell, Journal of the Society for Gynecologic Investigation 7:S10-S15 (2000), Goldstein, et al., Human Reproduction Update 6: 212-224(2000)]. The precise reason why the same compound can have cell-specificeffects has not been elucidated, but the differences in receptorconformation and/or in the milieu of coregulatory proteins have beensuggested.

[0008] It has been known for some time that estrogen receptors adoptdifferent conformations when binding ligands. However, the consequenceand subtlety of these changes has been only recently revealed. The threedimensional structures of ERα and ERβ have been solved byco-crystallization with various ligands and clearly show therepositioning of helix 12 in the presence of an estrogen receptorantagonist which sterically hinders the protein sequences required forreceptor-coregulatory protein interaction [Pike, et al., Embo 18:4608-4618 (1999), Shiau, et al., Cell 95: 927-937 (1998)]. In addition,the technique of phage display has been used to identify peptides thatinteract with estrogen receptors in the presence of different ligands[Paige, et al., Proceedings of the National Academy of Sciences of theUnited States of America 96: 3999-4004 (1999)]. For example, a peptidewas identified that distinguished between ERα bound to the full estrogenreceptor agonists 17β-estradiol and diethylstilbesterol. A differentpeptide was shown to distinguish between clomiphene bound to ERα andERβ. These data indicate that each ligand potentially places thereceptor in a unique and unpredictable conformation that is likely tohave distinct biological activities.

[0009] As mentioned above, estrogens affect a panoply of biologicalprocesses. In addition, where gender differences have been described(e.g. disease frequencies, responses to challenge, etc), it is possiblethat the explanation involves the difference in estrogen levels betweenmales and females.

DESCRIPTION OF THE INVENTION

[0010] This invention provides estrogenic compound of formula I havingthe structure,

[0011] wherein,

[0012] R¹ is phenyl optionally substituted with 1-4 Y groups;

[0013] R² is phenyl optionally substituted with 1-4 Y groups, alkyl of1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7carbon atoms, alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbonatoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,haloalkenyl of 2-7 carbon atoms, or haloalkynyl of 2-7 carbon atoms;

[0014] R³ is hydrogen, phenyl optionally substituted with 1-4 Y groups,alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of2-7 carbon atoms, alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbonatoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,haloalkenyl of 2-7 carbon atoms, or haloalkynyl of 2-7 carbon atoms;

[0015] X is O, —CH═CH—, or S;

[0016] Y is —OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbonatoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,perfluoroalkyl of 1-6 carbon atoms, or —COR⁴;

[0017] Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵, —NO₂, —CH═NR⁴, —CH═NOH,or —CH═NOR⁴;

[0018] R⁴ and R⁵ are each, independently, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or cycloalkylof 3-8 carbon atoms;

[0019] with the proviso that at least one of R² or R³ is phenyl orphenyl susbstituted with 1-4 Y groups;

[0020] or a pharmaceutically acceptable salt thereof.

[0021] Pharmaceutically acceptable salts can be formed from organic andinorganic acids, for example, acetic, propionic, lactic, citric,tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,camphorsulfonic, and similarly known acceptable aids when a compound ofthis invention contains a basic moiety. Salts may also be formed fromorganic and inorganic bases, such as alkali metal salts (for example,sodium, lithium, or potassium) alkaline earth metal salts, ammoniumsalts, alkylammonium salts containing 1-6 carbon atoms ordialkylammonium salts containing 1-6 carbon atoms in each alkyl group,and trialkylammonium salts containing 1-6 carbon atoms in each alkylgroup, when a compound of this invention contains an acidic moiety.

[0022] The terms alkyl, alkenyl, and alkynyl include both branched andstraight chain moieties. Examples include methyl, ethyl, propyl, butyl,isopropyl, sec-butyl, tert-butyl, vinyl, allyl, acetylene, 1-methylvinyl, and the like. The term halogen includes bromine, chlorine,fluorine, and iodine. The term alkylthio means -S-alkyl.

[0023] Examples of halogenated alkyl moieties include, but are notlimited to, chemical groups such as —CH₂F, —CH₂Cl, —CH₂Br, —CH₂CH₂F,—CH₂CH₂Cl, —CH₂CH₂Br, —CH₂CHF₂, —CH₂CHCl₂, —CH₂CHBr₂, —CH(CH₃)CH₂CH₂F,and higher homologs, isomers, and the like.

[0024] The term “halogenated alkenes”, employed alone, is defined hereinas, unless otherwise stated, either a straight chain or branched-chainmonovalent unsaturated hydrocarbon moiety, containing halogen atoms andat least one double bond. Examples of halogenated alkene moietiesinclude, but are not limited to, chemical groups such as —CH═CHF,—CH═CHCl, —CH═CHBr, —CH₂CH═CHF, —CH₂CH═CHBr, —CH═CHCHF, —CH═CHCHCl,—CH═CHCHBr, —C(CH₃)═CHF, —C(CH₃)═CHCl, —C(CH₃)═CHBr and higher homologs,isomers, and the like.

[0025] As used in accordance with this invention, the term “providing,”with respect to providing a compound or substance covered by thisinvention, means either directly administering such a compound orsubstance, or administering a prodrug, derivative, or analog which willform the effective amount of the compound or substance within the body.

[0026] Of the compounds of this invention, it is preferred that X is S.It is more preferred that X is S, R² is phenyl optionally substitutedwith 1-4 Y groups and R³ is hydrogen or alkyl of 1-6 carbon atoms. It isstill more preferred that X is S, R² is phenyl optionally substitutedwith 1-4 Y groups, R³ is hydrogen or alkyl of 1-6 carbon atoms, and Z is—CHO, —CN, —CH═NOH, or —CH═NOR⁴.

[0027] The reagents used in the preparation of the compounds of thisinvention can be either commercially obtained or can be prepared bystandard procedures described in the literature. The compounds of thepresent invention can be prepared according to the following syntheticSchemes (1-5).

[0028] The synthesis of two of the substituted phenylboronic acids usedin Suzuki couplings with halogenated thiophenes is outlined in Scheme 1.Treatment of the readily available 4-methoxy-2-trifluoromethyl anilinewith tert-butylnitrite and tetrafluoroboric acid in ethanol at 0° C.yielded the isolable diazonium salt. Bromination of the salt with CuBr₂in DMSO at room temperature gave the halide substituted aromatic.Lithium-halogen exchange with n-butyllithium in THF at −78° C., thenreaction with triisopropylborate and quenching with aqueous HCl yieldedthe required phenylboronic acid. The 3-chloro-4-methoxyboronic acid wassynthesized in a similar manner. 4-Bromo-3-chlorophenol was methylatedwith methyl iodide and potassium carbonate in refluxing acetone.Synthesis of the phenylboronic acid was achieved in the same manner asfor the 4-methoxy-2-trifluoromethylphenyl boronic acid.

[0029] Iodination of the commercially available3-bromo-4-methylthiophene with N-iodosuccinimide in chloroform-aceticacid yielded the di-iodinated thiophene (Scheme 2). Regiospecificlithium-halogen exchange with n-butyllithium in ether at −70° C. andquenching with DMF with an aqueous ammonium chloride work-up yielded the3-bromo-5-iodo-4-methylthiophene-2-carboxaldehyde. Protection of thecarboxaldehyde as the acetal was achieved with ethylene glycol inrefluxing benzene with p-toluenesulfonic acid. This2-(3-bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane was used as the oneof several precursors for the synthesis of key derivatives.

[0030] Preferential coupling of a substituted phenylboronic acid viaSuzuki coupling (Scheme 3) to2-(3-bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane under palladiumcatalysis, with base, yields the mono phenyl substituted thiophene. Thesecond coupling of a differentially substituted phenylboronic acid thenyields the diphenyl-substituted thiophene. Deprotection of the aldehydewith pyridinium-p-toluenesulfonic acid generates the aldehyde.Methoxy-substituted phenyl rings are then de-methylated with borontribromide to yield the corresponding phenols.

[0031] Differential substitution of the thiophene ring withphenylboronic acids was not always utilized (Scheme 4). In theseinstances the 3-bromo-5-iodo-4-methylthiophene-2-carboxaldehyde wasreacted with more than two equivalents of the substituted phenylboronicacid with a palladium catalyst. The resulting diphenyl-substitutedthiophene containing methoxy-substituted phenyl rings were demethylatedwith boron tribromide to yield the corresponding phenols.

[0032] The synthesis of R-substituted oximes and compounds containing anitrile function were synthesized from the thiophene-2-aldehyde asdescribed in Scheme 5. A diphenyl-substituted thiophene carboxaldehydewas reacted with a hydroxylamine-hydrochloride in pyridine and ethanolto yield the oxime. Demethylation of the methoxy groups with borontribromide provided the phenol-substituted thiophene. The oxime was thendehydrated to the nitrile with pyridinium hydrobromide.

[0033] Standard pharmacological test procedures are readily available todetermine the activity profile of a given test compound. The followingbriefly summarizes several representative test procedures and mayinclude data for representative compounds of the invention. All assays,except the radioligand binding assay, can be used to detect estrogenreceptor agonist or antagonist activity of compounds. In general,estrogen receptor agonist activity is measured by comparing the activityof the compound to a reference estrogen (e.g. 17β-estradiol,17α-ethinyl, 17β-estradiol, estrone, diethylstilbesterol etc). Estrogenreceptor antagonist activity is generally measured by co-treating thetest compound with the reference estrogen and comparing the result tothat obtained with the reference estrogen alone. Standardpharmacological test procedures for SERMs are also provided in U.S. Pat.Nos. 4,418,068 and 5,998,402 which are hereby incorporated by reference.

[0034] Evaluation of Binding Affinities to ERα and ERβ

[0035] Representative examples of the invention were evaluated for theirability to compete with 17β-estradiol for both ERα and ERβ in aconventional radioligand binding assay. This test procedure provides themethodology for one to determine the relative binding affinities for theERα or ERβ recptors. The procedure used is briefly described below.

[0036] Preparation of receptor extracts for characterization of bindingselectivity. The ligand binding domains, conveniently defined here asall sequence downstream of the DNA binding domain, were obtained by PCRusing full length cDNA as templates and primers that containedappropriate restriction sites for subcloning while maintaining theappropriate reading frame for expression. These templates containedamino acids M₂₅₀-V₅₉₅ of human ERα [Green, et al., Nature 320: 134-9(1986)] and M₂₁₄-Q₅₃₀ of human ERβ [Ogawa, et al., Biochemical &Biophysical Research Communications 243: 122-6 (1998)]. Human ERβ wascloned into pET15b (Novagen, Madison Wis.) as a Nco1-BamH1 fragmentbearing a C-terminal Flag tag. Human ERα was cloned as for human ERβexcept that an N-terminal His tag was added. The sequences of allconstructs used were verified by complete sequencing of both strands.

[0037] BL21(DE3) cells were used to express the human proteins.Typically a 10 mL overnight culture was used to inoculate a 1 L cultureof LB medium containing 100 μg/mL of ampicillin. After incubationovernight at 37° C., IPTG was added to a final concentration of 1 mM andincubation proceeded at 25° C. for 2 hours. Cells were harvested bycentrifugation (1500×g), and the pellets washed with and resuspended in100 mL of 50 mM Tris-Cl (pH 7.4), 150 mM NaCl. Cells were lysed bypassing twice through a French press at 12000 psi. The lysate wasclarified by centrifugation at 12,000×g for 30 minutes at 4° C. andstored at −70° C.

[0038] Evaluation of extracts for specific [³H]-estradiol binding.Dulbecco's phosphate buffered saline (Gibco, 1× final concentration)supplemented with 1 mM EDTA was used as the assay buffer. To optimizethe amount of receptor to use in the assay, [³H]-17β-estradiol (NewEngland Nuclear; final concentration=2 nM)±0.6 μM diethlystilbestrol and100 μL of various dilutions of the E. coli lysate were added to eachwell of a high binding masked microtiter plate (EG&G Wallac). The finalassay volume was 120 μL and the concentration of DMSO was ≦1%. Afterincubation at room temperature for 5-18 hours, unbound material wasaspirated and the plate washed three times with approximately 300 μL ofassay buffer. After washing, 135 μL of scintillation cocktail (OptiphaseSupermix, EG&G Wallac) was added to the wells, and the plate was sealedand agitated for at least 5 minutes to mix scintillant with residualwash buffer. Bound radioactivity was evaluated by liquid scintillationcounting (EG&G Wallac Microbeta Plus).

[0039] After determining the dilution of each receptor preparation thatprovided maximum specific binding, the assay was further optimized byestimating the IC₅₀ of unlabelled 17β-estradiol using various dilutionsof the receptor preparation. A final working dilution for each receptorpreparation was chosen for which the IC₅₀ of unlabelled 17β-estradiolwas 2-4 nM.

[0040] Ligand binding competition test procedure. Test compounds wereinitially solubilized in DMSO and the final concentration of DMSO in thebinding assay was ≦1%. Eight dilutions of each test compound were usedas an unlabelled competitor for [³H]-17β-estradiol. Typically, a set ofcompound dilutions would be tested simultaneously on human ERα and ERβ.The results were plotted as measured DPM vs. concentration of testcompound. For dose-response curve fitting, a four parameter logisticmodel on the transformed, weighted data was fit and the IC₅₀ was definedas the concentration of compound decreasing maximum [³H]-estradiolbinding by 50%.

[0041] Binding affinities for ERα and ERβ (as measured by IC₅₀) forrepresentative examples of the invention are shown in Table (1). TABLE 1Estrogen receptor binding affinities of compounds of the inventionExample ER-β IC₅₀ (μM) ER-α IC₅₀ (μM) 1 0.054 0.212 2 0.113 0.394 30.0055 0.029 4 0.174 0.464 5 0.0018 0.0355 6 0.071 0.515 7 0.0017 0.0318 0.0053 0.093 9 0.012 0.085 10 0.004 0.088 11 0.0078 0.068 12 0.0130.089 13 0.027 0.293 14 0.016 0.157 15 0.0021 0.031 16 0.091 0.275 170.083 0.305 18 0.042 0.086 19 0.0036 0.04 20 0.028 0.152 21 0.123 0.19622 0.073 0.299 23 0.057 0.073

[0042] The results obtained in the standard pharmacologic test proceduredescribed above demonstrate that the compounds of this invention bindboth subtypes of the estrogen receptor. The IC₅₀s are generally lowerfor ERβ, indicating these compounds are preferentially ERβ selectiveligands, but are still considered active at ERα. Compounds of thisinvention will exhibit a range of activity based, at least partially, ontheir receptor affinity selectivity profiles. Since the compounds of theinvention bind ER-β with higher affinity than ER-α, they will be usefulin treating or inhibiting diseases that can be modulated via ER-β.Additionally, since each receptor ligand complex is unique and thus itsinteraction with various coregulatory proteins is unique, compounds ofthis invention will display different and unpredictable activitiesdepending on cellular context. For example, in some cell-types, it ispossible for a compound to behave as an estrogen receptor agonist whilein other tissues, an estrogen receptor antagonist. Compounds with suchactivity have sometimes been referred to as SERMs (Selective EstrogenReceptor Modulators). Unlike many estrogens, however, many of the SERMsdo not cause increases in uterine wet weight. These compounds areantiestrogenic in the uterus and can completely antagonize the trophiceffects of estrogen receptor agonists in uterine tissue. Thesecompounds, however, act as estrogen receptor agonists in the bone,cardiovascular, and central nervous systems. Due to this tissueselective nature of these compounds, they are useful in treating orinhibiting in a mammal disease states or syndromes which are caused orassociated with an estrogen deficiency (in certain tissues such as boneor cardiovascular) or an excess of estrogen (in the uterus or mammaryglands). In addition, compounds of this invention also have thepotential to behave as estrogen receptor agonists on one receptor typewhile behaving as estrogen receptor antagonists on the other. Forexample, it has been demonstrated that compounds can be antagonize theaction of 17β-estradiol via ERβ while exhibiting estrogen receptoragonist activity with ERα [Sun, et al., Endocrinology 140: 800-804(1999)]. Such ERSAA (Estrogen Receptor Selective Agonist Antagonist)activity provides for pharmacologically distinct estrogenic activitywithin this series of compounds

[0043] Regulation of Metallothionein II mRNA

[0044] Estrogens acting through ERβ, but not ERα can upregulatemetallothionein II mRNA levels in Saos-2 cells as described by Harris[Endocrinology 142: 645-652 (2001)]. Results from this test procedurecan be combined with results from the test procedure described below(ERE reporter test procedure) to generate a selectivity profile forcompounds of this invention (see also WO 00/37681). Data forrepresentative compounds of the invention are shown in Table (2). TABLE2 Upregulation of metallothionein-II mRNA in Saos-2 cells by selectedcompounds of the invention Example Fold upregulation 15 6.0

[0045] Evaluation of Test Compound Using an ERE-Reporter Test Procedurein MCF-7 Breast Cancer Cells

[0046] Stock solutions of test compounds (usually 0.1 M) are prepared inDMSO and then diluted 10 to 100-fold with DMSO to make working solutionsof 1 or 10 mM. The DMSO stocks are stored at either 4° C. (0.1 M) or−20° C. (<0.1M). MCF-7 cells are passaged twice a week with growthmedium [D-MEM/F-12 medium containing 10% (v/v) heat-inactivated fetalbovine serum, 1% (v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1].The cells are maintained in vented flasks at 37° C. inside a 5% CO₂/95%humidified air incubator. One day prior to treatment, the cells areplated with growth medium at 25,000 cells/well into 96 well plates andincubated at 37° C. overnight.

[0047] The cells are infected for 2 hr at 37° C. with 50 μl/well of a1:10 dilution of adenovirus 5-ERE-tk-luciferase in experimental medium[phenol red-free D-MEM/F-12 medium containing 10% (v/v) heat-inactivedcharcoal-stripped fetal bovine serum, 1% (v/v) Penicillin-Streptomycin,2 mM glutaMax-1, 1 mM sodium pyruvate]. The wells are then washed oncewith 150 μl of experimental medium. Finally, the cells are treated for24 hr at 37° C. in replicates of 8 wells/treatment with 150 μl/well ofvehicle (≦0.1% v/v DMSO) or compound that is diluted≧1000-fold intoexperimental medium.

[0048] Initial screening of test compounds is done at a single dose of 1μM that is tested alone (estrogen receptor agonist mode) or incombination with 0.1 nM 17β-estradiol (EC₈₀; estrogen receptorantagonist mode). Each 96 well plate also includes a vehicle controlgroup (0.1% v/v DMSO) and an estrogen receptor agonist control group(either 0.1 or 1 nM 17β-estradiol). Dose-response experiments areperformed in either the estrogen receptor agonist and/or estrogenreceptor antagonist modes on active compounds in log increases from10⁻¹⁴ to 10⁻⁵ M. From these dose-response curves, EC₅₀ and IC₅₀ values,respectively, are generated. The final well in each treatment groupcontains 5 μl of 3×10⁻⁵ M ICI-182,780 (10⁻⁶ M final concentration) as anestrogen receptor antagonist control.

[0049] After treatment, the cells are lysed on a shaker for 15 min with25 μl/well of 1× cell culture lysis reagent (Promega Corporation). Thecell lysates (20 μl) are transferred to a 96 well luminometer plate, andluciferase activity is measured in a MicroLumat LB 96 P luminometer (EG& G Berthold) using 100 μl/well of luciferase substrate (PromegaCorporation). Prior to the injection of substrate, a 1 second backgroundmeasurement is made for each well. Following the injection of substrate,luciferase activity is measured for 10 seconds after a 1 second delay.The data are transferred from the luminometer to a Macintosh personalcomputer and analyzed using the JMP software (SAS Institute); thisprogram subtracts the background reading from the luciferase measurementfor each well and then determines the mean and standard deviation ofeach treatment.

[0050] The luciferase data are transformed by logarithms, and the HuberM-estimator is used to down-weight the outlying transformedobservations. The JMP software is used to analyze the transformed andweighted data for one-way ANOVA (Dunnett's test). The compoundtreatments are compared to the vehicle control results in the estrogenreceptor agonist mode, or the positive estrogen receptor agonist controlresults (0.1 nM 17β-estradiol) in the estrogen receptor antagonist mode.For the initial single dose experiment, if the compound treatmentresults are significantly different from the appropriate control(p<0.05), then the results are reported as the percent relative to the17β-estradiol control [i.e., ((compound−vehicle control)/(17β-estradiolcontrol−vehicle control))×100]. The JMP software is also used todetermine the EC₅₀ and/or IC₅₀ values from the non-linear dose-responsecurves.

[0051] Evaluation of Uterotrophic Activity

[0052] Uterotrophic activity of a test compound can be measuredaccording to the following standard pharmacological test procedures.

[0053] Procedure 1: Sexually immature (18 days of age) Sprague-Dawleyrats are obtained from Taconic and provided unrestricted access to acasein-based diet (Purina Mills 5K96C) and water. On day 19, 20 and 21the rats are dosed subcutaneously with 17α-ethinyl-17β-estradiol (0.06μg/rat/day), test compound or vehicle (50% DMSO/50% Dulbecco's PBS). Toassess estrogen receptor antagonist, compounds are coadministered with17α-ethinyl-17β-estradiol (0.06 μg/rat/day). There are six rats/groupand they are euthanized approximately 24 hours after the last injectionby CO₂ asphyxiation and pneumothorax. Uteri are removed and weighedafter trimming associated fat and expressing any internal fluid. Atissue sample can also be snap frozen for analysis of gene expression(e.g. complement factor 3 mRNA).

[0054] Procedure 2: Sexually immature (18 days of age) 129 SvE mice areobtained from Taconic and provided unrestricted access to a casein-baseddiet (Purina Mills 5K96C) and water. On day 22, 23, 24 and 25 the miceare dosed subcutaneously with compound or vehicle (corn oil). There aresix mice/group and they are euthanized approximately 6 hours after thelast injection by CO₂ asphyxiation and pneumothorax. Uteri are removedand weighed after trimming associated fat and expressing any internalfluid.

[0055] Evaluation of Osteoporosis and Lipid Modulation(Cardioprotection)

[0056] Female Sprague-Dawley rats, ovariectomized or sham operated, areobtained 1 day after surgery from Taconic Farms (weight range 240-275g). They are housed 3 or 4 rats/cage in a room on a 12/12 (light/dark)schedule and provided with food (Purina 5K96C rat chow) and water adlibitum. Treatment for all studies begin 1 day after arrival and ratsare dosed 7 days per week as indicated for 6 weeks. A group of agematched sham operated rats not receiving any treatment serve as anintact, estrogen replete control group for each study.

[0057] All test compounds are prepared in a vehicle of 50% DMSO (JTBaker, Phillipsburg, N.J.)/1× Dulbecco's phosphate saline (GibcoBRL,Grand Island, N.Y.) at defined concentrations so that the treatmentvolume is 0.1 mL/100 g body weight. 17β-estradiol is dissolved in cornoil (20 μg/mL) and delivered subcutaneously, 0.1 mL/rat. All dosages areadjusted at three week intervals according to group mean body weightmeasurements, and given subcutaneously.

[0058] Five weeks after the initiation of treatment and one week priorto the termination of the study, each rat is evaluated for bone mineraldensity (BMD). The total and trabecular density of the proximal tibiaare evaluated in anesthetized rats using an XCT-960M (pQCT; StratecMedizintechnik, Pforzheim, Germany). The measurements are performed asfollows: Fifteen minutes prior to scanning, each rat is anesthetizedwith an intraperitoneal injection of 45 mg/kg ketamine, 8.5 mg/kgxylazine, and 1.5 mg/kg acepromazine.

[0059] The right hind limb is passed through a polycarbonate tube with adiameter of 25 mm and taped to an acrylic frame with the ankle joint ata 90° angle and the knee joint at 1800. The polycarbonate tube isaffixed to a sliding platform that maintains it perpendicular to theaperture of the pQCT. The platform is adjusted so that the distal end ofthe femur and the proximal end of the tibia is in the scanning field. Atwo dimensional scout view is run for a length of 10 mm and a lineresolution of 0.2 mm. After the scout view is displayed on the monitor,the proximal end of the tibia is located. The pQCT scan is initiated 3.4mm distal from this point. The pQCT scan is 1 mm thick, has a voxel(three dimensional pixel) size of 0.140 mm, and consists of 145projections through the slice.

[0060] After the pQCT scan is completed, the image is displayed on themonitor. A region of interest including the tibia but excluding thefibula is outlined. The soft tissue is mathematically removed using aniterative algorithm. The density of the remaining bone (total density)is reported in mg/cm³. The outer 55% of the bone is mathematicallypeeled away in a concentric spiral. The density of the remaining bone(Trabecular density) is reported in mg/cm³.

[0061] One week after BMD evaluation the rats are euthanized by CO₂asphyxiation and pneumothorax, and blood is collected for cholesteroldetermination. The uteri are also removed and the weighed after trimmingassociated fat and expressing any luminal fluid. Total cholesterol isdetermined using a Boehringer-Mannheim Hitachi 911 clinical analyzerusing the Cholesterol/HP kit. Statistics were compared using one-wayanalysis of variance with Dunnet's test.

[0062] Evaluation of Antioxidant Activity

[0063] Porcine aortas are obtained from an abattoir, washed, transportedin chilled PBS, and aortic endothelial cells are harvested. To harvestthe cells, the intercostal vessels of the aorta are tied off and one endof the aorta clamped. Fresh, sterile filtered, 0.2% collagenase (SigmaType I) is placed in the vessel and the other end of the vessel thenclamped to form a closed system. The aorta is incubated at 37° C. for15-20 minutes, after which the collagenase solution is collected andcentrifuged for 5 minutes at 2000×g. Each pellet is suspended in 7 mL ofendothelial cell culture medium consisting of phenol red free DMEM/Ham'sF12 media supplemented with charcoal stripped FBS (5%), NuSerum (5%),L-glutamine (4 mM), penicillin-streptomycin (1000 U/ml, 100 μg/ml) andgentamycin (75 μg/ml), seeded in 100 mm petri dish and incubated at 37°C. in 5%CO₂. After 20 minutes, the cells are rinsed with PBS and freshmedium added, this was repeated again at 24 hours. The cells areconfluent after approximately 1 week. The endothelial cells areroutinely fed twice a week and, when confluent, trypsinized and seededat a 1:7 ratio. Cell mediated oxidation of 12.5 μg/mL LDL is allowed toproceed in the presence of the compound to be evaluated (5 μM) for 4hours at 37° C. Results are expressed as the percent inhibition of theoxidative process as measured by the TBARS (thiobarbituric acid reactivesubstances) method for analysis of free aldehydes [Yagi, BiochemicalMedicine 15: 212-6 (1976)].

[0064] Progesterone Receptor mRNA Regulation Standard PharmacologicalTest Procedure

[0065] This test procedure can be used to evaluate the estrogenic orantiestrogenic activity of compounds from this invention [Shughrue, etal., Endocrinology 138: 5476-5484 (1997)].

[0066] Rat Hot Flush Test Procedure

[0067] The effect of test compounds on hot flushes can be evaluated in astandard pharmacological test procedure which measures the ability of atest compound to blunt the increase in tail skin temperature whichoccurs as morphine-addicted rats are acutely withdrawn from the drugusing naloxone [Merchenthaler, et al., Maturitas 30: 307-16 (1998)]. Itcan also be used to detect estrogen receptor antagonist activity byco-dosing test compound with the reference estrogen.

[0068] Evaluation of Vasomotor Function in Isolated Rat Aortic Rings

[0069] Sprague-Dawley rats (240-260 grams) are divided into 4 groups:

[0070] 1. Normal non-ovariectomized (intact)

[0071] 2. Ovariectomized (ovex) vehicle treated

[0072] 3. Ovariectomized 17β-estradiol treated (1 mg/kg/day)

[0073] 4. Ovariectomized animals treated with test compound (variousdoses)

[0074] Animals are ovariectomized approximately 3 weeks prior totreatment. Each animal receives either 17-β estradiol sulfate (1mg/kg/day) or test compound suspended in distilled, deionized water with1% tween-80 by gastric gavage. Vehicle treated animals received anappropriate volume of the vehicle used in the drug treated groups.

[0075] Animals are euthanized by CO₂ inhalation and exsanguination.Thoracic aortae are rapidly removed and placed in 37° C. physiologicalsolution with the following composition (mM): NaCl (54.7), KCl (5.0),NaHCO₃ (25.0), MgCl₂ 2H₂O (2.5), D-glucose (11.8) and CaCl₂ (0.2) gassedwith CO₂—O₂, 95%/5% for a final pH of 7.4. The advantitia is removedfrom the outer surface and the vessel is cut into 2-3 mm wide rings.Rings are suspended in a 10 mL tissue bath with one end attached to thebottom of the bath and the other to a force transducer. A restingtension of 1 gram is placed on the rings. Rings are equilibrated for 1hour, signals are acquired and analyzed.

[0076] After equilibration, the rings are exposed to increasingconcentrations of phenylephrine (10⁻⁸ to 10⁻⁴ M) and the tensionrecorded. Baths are then rinsed 3 times with fresh buffer. Afterwashout, 200 mM L-NAME is added to the tissue bath and equilibrated for30 minutes. The phenylephrine concentration response curve is thenrepeated.

[0077] Evaluation of Cardioprotective Activity

[0078] Apolipoprotein E-deficient C57/B1J (apo E KO) mice are obtainedfrom Taconic Farms. All animal procedures are performed under strictcompliance to IACUC guidelines. Ovariectomized female apo E KO mice, 4-7weeks of age, are housed in shoe-box cages and were allowed free accessto food and water. The animals are randomized by weight into groups(n=12-15 mice per group). The animals are dosed with test compounds orestrogen (17β-estradiol sulfate at 1 mg/kg/day) in the diet using aPrecise-dosing Protocol, where the amount of diet consumed is measuredweekly, and the dose adjusted accordingly, based on animal weight. Thediet used is a Western-style diet (57U5) that is prepared by Purina andcontains 0.50% cholesterol, 20% lard and 25 IU/KG Vitamin E. The animalsare dosed/fed using this paradigm for a period of 12 weeks. Controlanimals are fed the Western-style diet and receive no compound. At theend of the study period, the animals are euthanized and plasma samplesobtained. The hearts are perfused in situ, first with saline and thenwith neutral buffered 10% formalin solution.

[0079] For the determination of plasma lipids and lipoproteins, totalcholesterol and triglycerides are determined using enzymatic methodswith commercially available kits from Boehringer Mannheim and WakoBiochemicals, respectively and analyzed using the Boehringer MannheimHitachii 911 Analyzer. Separation and quantification of plasmalipoproteins were performed using FPLC size fractionation. Briefly,50-100 mL of serum is filtered and injected into Superose 12 andSuperose 6 columns connected in series and eluted at a constant flowrate with 1 mM sodium EDTA and 0.15 M NaCl. Areas of each curverepresenting VLDL, LDL and HDL are integrated using Waters Millennium™software, and each lipoprotein fraction is quantified by multiplying theTotal Cholesterol value by the relative percent area of each respectivechromatogram peak.

[0080] For the quantification of aortic atherosclerosis, the aortas arecarefully isolated and placed in formalin fixative for 48-72 hoursbefore handling. Atherosclerotic lesions are identified using Oil Red Ostaining. The vessels are briefly destained, and then imaged using aNikon SMU800 microscope fitted with a Sony 3CCD video camera system inconcert with IMAQ Configuration Utility (National Instrument) as theimage capturing software. The lesions are quantified en face along theaortic arch using a custom threshold utility software package (ColemanTechnologies). Automated lesion assessment is performed on the vesselsusing the threshold function of the program, specifically on the regioncontained within the aortic arch from the proximal edge of thebrachio-cephalic trunk to the distal edge of the left subclavian artery.Aortic atherosclerosis data are expressed as percent lesion involvementstrictly within this defined luminal area.

[0081] Evaluation of Cognition Enhancement

[0082] Ovariectomized rats (n=50) are habituated to an 8-arm radial armmaze for 10-min periods on each of 5 consecutive days. Animals arewater-deprived prior to habituation and testing. A 100 mL aliquot ofwater placed at the ends of each arm serves as reinforcement.Acquisition of a win-shift task in the radial arm maze is accomplishedby allowing the animal to have access to one baited arm. After drinking,the animal exits the arm and re-enters the central compartment, where itnow has access to the previously visited arm or to a novel arm. Acorrect response is recorded when the animal chooses to enter a novelarm. Each animal is given 5 trials per day for 3 days. After the lastacquisition trial, the animals are assigned to one of the following 4groups:

[0083] 1. Negative controls: injected with 10% DMSO/ sesame oil vehicleonce daily for 6 days (1 mUkg, SC)

[0084] 2. Positive controls: injected with 17β-estradiol benzoate for 2days and tested 4 days after the second injection (17β-estradiolbenzoate at 10 μg/0.1 mL per rat)

[0085] 3. Estradiol: 17β-estradiol will be injected daily for 6 days (20μg/kg, SC)

[0086] 4. Test compound: injected daily for 6 days (doses vary).

[0087] All injections will begin after testing on the last day ofacquisition. The last injection for groups 1, 3, and 4 will take place 2hours before testing for working memory.

[0088] The test for working memory is a delayed non-matching-to-sampletask (DNMS) utilizing delays of 15, 30, or 60 seconds. This task is avariation of the acquisition task in which the rat is placed in thecentral arena and allowed to enter one arm as before. A second arm isopened once the rat traverses halfway down the first arm, and again therat is required to choose this arm. When it has traveled halfway downthis second arm, both doors are closed and the delay is instituted. Oncethe delay has expired, both of the original two doors, and a third noveldoor, are opened simultaneously. A correct response is recorded when theanimal travels halfway down the third, novel arm. An incorrect responseis recorded when the animal travels halfway down either the first orsecond arms. Each animal will receive 5 trials at each of the threedelay intervals for a total of 15 trials per subject.

[0089] Evaluation of Effect on Pleurisy

[0090] The ability to reduce the symptoms of experimentally-inducedpleurisy in rats can be evaluated according to the procedure ofCuzzocrea [Endocrinology 141: 1455-63 (2000)].

[0091] Evaluation of Protection Against Glutamate-Induced Cytotoxicity(Neuroprotection)

[0092] The neuroprotective activity of compounds of this invention canbe evaluated in an in vitro standard pharmacological test procedureusing glutamate challenge [Zaulyanov, et al., Cellular & MolecularNeurobiology 19: 705-18 (1999); Prokai, et al., Journal of MedicinalChemistry 44: 110-4 (2001)].

[0093] Evaluation in the Mammary End Bud Test Procedure

[0094] Estrogens are required for full ductal elongation and branchingof the mammary ducts, and the subsequent development of lobulo-alveolarend buds under the influence of progesterone. In this test procedure,the mammotrophic activity of selected compounds of the invention can beevaluated according to the following standard pharmacological testprocedure. Twenty-eight day old Sprague-Dawley rats (Taconic Farms,Germantown, N.Y.) are ovariectomized and rested for nine days. Animalsare housed under a 12-hour light/dark cycle, fed a casein-based PurinaLaboratory Rodent Diet 5K96 (Purina, Richmond, Ind.) and allowed freeaccess to water. Rats were then dosed subcutaneously for six days withvehicle (50% DMSO (J T Baker, Phillipsburg, N.J.)/50% 1× Dulbecco'sPhosphate buffered saline (GibcoBRL, Grand Island, N.Y.), 17β-estradiol(0.1 mg/kg) or test compound (20 mg/kg). For the final three days, ratsare also dosed subcutaneously with progesterone (30 mg/kg). On theseventh day, rats are euthanised and a mammary fat pad excised. This fatpad is analyzed for casein kinase II mRNA as a marker of end budproliferation. Casein kinase II mRNA is anlayzed by real-time RT-PCR.Briefly, RNA is isolated following Trizol (GibcoBRL, Grand Island, N.Y.)according to the manufacture's directions, Samples are treated withDNAse I using DNA-free kit (Ambion), and casein kinase II mRNA levelsare measured by real-time RT-PCR using the Taqman Gold procedure (PEApplied Biosystems). A total of 50 ng of RNA is analyzed in triplicateusing casein kinase II specific primer pair (5′ primer,CACACGGATGGCGCATACT; 3′ primer, CTCGGGATGCACCATGAAG) and customizedprobe (TAMRA-CGGCACTGGTTTCCCTCACATGCT-FAM). Casein kinase II mRNA levelsare normalized to 18s ribosomal RNA contained within each samplereaction using primers and probe supplied by PE Applied Biosystems.

[0095] Evaluation in the HLA Rat Standard Pharmacological Test Procedurefor Inflammatory Bowel Disease

[0096] Representative compounds can be evaluated in the HLA rat standardpharmacological test procedure which emulates inflammatory bowel diseasein humans. The following briefly describes the procedure used andresults obtained. Male HLA-B27 rats are obtained from Taconic andprovided unrestricted access to food (PMI Lab diet 5001) and water. Ratsare dosed subcutaneously once per day with either vehicle (50% DMSO/50%1× Dulbecco's Phosphate Buffered Saline) or test compound (0.1 to 10mg/kg) for at least one week. Stool quality is observed daily and gradedaccording to the following scale: Diarrhea=3; soft stool=2; normalstool=1. At the end of the study, serum is collected and stored at −70°C. A section of colon is prepared for histological analysis and anadditional segment is analyzed for myeloperoxidase activity.

[0097] For histological analysis, colonic tissue is immersed in 10%neutral buffered formalin. Each specimen of colon is separated into foursamples for evaluation. The formalin-fixed tissues are processed in aTissue Tek vacuum infiltration processor (Miles, Inc; West Haven, Conn.)for paraffin embedding. The samples are sectioned at 5 μm and thenstained with hematoxylin and eosin (H&E) for blinded histologicevaluations using a scale modified after Boughton-Smith. After thescores are completed the samples are unblinded, and data are tabulatedand analyzed by ANOVA linear modeling with multiple mean comparisons.Sections of colonic tissue are evaluated for several disease indicatorsand given relative scores.

[0098] Evaluation in Two Models of Arthritis

[0099] Lewis rat assay of adjuvant-induced arthritis. Sixty, female, 12weeks old, Lewis rats are housed according to standard facilityoperating procedures. They receive a standard regimen of food and waterad libitum. Each animal is identified by a cage card indicating theproject group and animal number. Each rat number is marked by indelibleink marker on the tail. At least 10-21 days before study they areanesthetized and ovariectomized by standard aseptic surgical techniques.

[0100] Freund's Adjuvant-Complete (Sigma Immuno Chemicals, St. Louis,Mo.) is used to induce arthritis, each mL containing 1 mg Mycobacteriumtuberculosis heat killed and dried, 0.85 mL mineral oil and 0.15 mLmannide monooleate Lot No. 084H8800.

[0101] The following are examples of two test procedures. Inhibitiontest procedure: Thirty rats are injected intradermally with 0.1 mL ofFreund's Adjuvant-Complete at the base of the tail. The animals arerandomized to four groups, each group containing six rats. Each day, thegroups receive vehicle (50% DMSO (JT Baker, Phillipsburg, N.J.)/ 1×Dulbecco's phosphate saline (GibcoBRL, Grand Island, N.Y.)) or testcompound (0.1-10 mg/kg, administered subcutaneously). All rats begintreatment on Day 1.

[0102] Treatment test procedure: Thirty rats are injected intradermallywith 0.1 mL of Freund's Adjuvant-Complete at the base of the tail. Theanimals are randomized to four groups, each group containing six rats.Each day, the groups receive vehicle (50% DMSO (JT Baker, Phillipsburg,N.J.)/1× Dulbecco's phosphate saline (GibcoBRL, Grand Island, N.Y.)) ortest compound (0.1-10 mg/kg, administered subcutaneously). All ratsbegin treatment on Day 8 after adjuvant injection.

[0103] Statistical analysis is performed using Abacus Concepts SuperANOVA. (Abacus Concepts, Inc., Berkeley, Calif.). All of the parametersof interest are subjected to Analysis of Variance with Duncan's newmultiple range post hoc testing between groups. Data are expressedthroughout as mean±standard deviation (SD), and differences are deemedsignificant if p<0.05.

[0104] The degree of arthritis severity is monitored daily in terms ofthe following disease indices: Hindpaw erythema, hindpaw swelling,tenderness of the joints, and movements and posture. An integer scale of0 to 3 is used to quantify the level of erythema (0=normal paw, 1=milderythema, 2=moderate erythema, 3=severe erythema) and swelling (0=normalpaw, 1=mild swelling, 2=moderate swelling, 3=severe swelling of the hindpaw). The maximal score per day is 12.

[0105] At the end of the study the rats are euthanized with CO₂,hindlimbs removed at necropsy and fixed in 10% buffered formalin, andthe tarsal joints decalcified and embedded in paraffin. Histologicsections are stained with Hematoxylin and Eosin or Saffranin O—FastGreen stain.

[0106] Slides are coded so that the examiner is blinded to the treatmentgroups. Synovial tissue from tarsal joints is evaluated based onsynovial hyperplasia, inflammatory cell infiltration, and pannusformation [Poole and Coombs, International Archives of Allergy & AppliedImmunology 54: 97-113 (1977)] as outlined below. Category Grade 1.Synovial lining cells  a. No change 0  b. Cells enlarged, slightlythickened 1  c. Cells enlarged, increase in numbers, moderatelythickened. 2  No villus present  d. Cells enlarged, thickened. Villuspresent 3 2. Fibroplasia  a. No change 0  b. Fibroplasia present underlining cells 1  c. Small areas of areolar tissue replaced by fibroustissue 2  d. Replacement of areolar tissue by fibrous tissue 3 3.Inflammatory cells  a. Occasionally seen, scattered throughout selection0  b. Cells present in small numbers in or just under lining 1  celllayer and/or around blood vessels.  c. Small focal collection of cellsmay be present 2  d. Large numbers of cells present in capsule and in 3 or under lining cell layers. Large foci often seen. 4. Pannus  a. Notdetectable 0  b. Detectable 1

[0107] In addition, articular cartilage and bone is evaluated usingMankin's histological grading system [Mankin, et al., Journal of Bone &Joint Surgery—American Volume 53: 523-37 (1971)] as shown below.Category Grade 1. Structure  a. Normal 0  b. Surface irregularity 1  c.Pannus and surface irregularity 2  d. Clefts to transitional zone 3  e.Clefts to radial zone 4  f. Clefts to calcified zone 5  g. Completedisorganization 6 2. Cells  a. Normal 0  b. Diffuse hypercellularity 1 c. Cloning 2  d. Hypocellularity 3 3. Safranin-O staining  a. Normal 0 b. Slight reduction 1  c. Modest reduction 2  d. Severe reduction 3  e.No dye noted 4 4. Tidemark integrity  a. Intact 0  b. Crossed by bloodvessels 1

[0108] Evaluation in the HLA-B27 Rat model of arthritis. Representativecompounds are evaluated in the HLA-B27 rat standard pharmacological testprocedure which emulates arthritis in humans. The following brieflydescribes the procedure used. Male HLA-B27 rats are obtained fromTaconic and provided unrestricted access to a food (PMI Lab diet 5001)and water. Rats are dosed subcutaneously once per day with eithervehicle (50% DMSO/50% 11× Dulbecco's Phosphate Buffered Saline) or testcompound (0.1 to 10 mg/kg) for at least one week. Joint scores andhistology are evaluated as described above for the Lewis rat model ofadjuvant-induced arthritis.

[0109] Evaluation in in vivo Models of Carcinogeneisis

[0110] The ability of compounds of this invention to treat and inhibitvarious malignancies or hyperprolific disorders can be evaluated instandard pharmacological test procedures that are readily available inthe literature, and include the following two procedures.

[0111] Breast cancer. Athymic nu/nu (nude) mice are obtainedovariectomized from Charles River Laboratories (Wilmington, Mass.). Oneday prior to tumor cell injection, animals are implanted withtime-release pellets containing 0.36-1.7 mg 17β-estradiol (60 or 90 dayrelease, Innovative Research of America, Sarasota, Fla.) or a placebo.The pellet is introduced subcutaneously into the intrascapular regionusing a 10-gauge precision trochar. Subsequently, mice are injectedsubcutaneously into the breast tissue with either 1×10⁷ MCF-7 cells or1×10⁷ BG-1 cells. The cells are mixed with an equal volume of matrigel,a basement membrane matrix preparation to enhance tumor establishment.Test compounds can be evaluated either by dosing one day after tumorcell implantation (inhibition regimen) or after tumors have reached acertain size (treatment regimen). Compounds are administered eitherintraperitoneally or orally in a vehicle of 1% tween-80 in saline eachday. Tumor size is evaluated every three or seven days.

[0112] Colon cancer. The ability to treat or inhibit colon cancer can beevaluated in the test procedure of Smirnoff [Oncology Research 11:255-64 (1999)].

[0113] Evaluation of Neuroprotection in Two in vivo Test Procedures

[0114] Transient global ischemia in the Mongolian gerbil. The effect oftest compounds on preventing or treating brain injury in response tooxygen deprivation/reperfusion can be measured using the following testprocedure.

[0115] Female Mongolian gerbils (60-80 g; Charles River Laboratories,Kingston, N.Y.) are housed in the Wyeth-Ayerst animal care facility(AAALAC certified) with a 12-hour light, 12-hour dark photoperiod andfree access to tap water and a low-estrogen casein diet (Purina;Richmond, Ind.). After acclimation (3-5 days), gerbils are anesthetizedwith isoflurane (2-3% mixture with O₂), ovariectomized (Day 0).Beginning the following morning (Day 1), gerbils are treatedsubcutaneously each day with either vehicle (10% ETOH/corn oil),17β-estradiol (1 mg/kg) or an experimental compound (0.1-20 mg/kg). OnDay 6, gerbils (n=4-5/group) are anesthetized with isoflurane, thecommon carotid arteries visualized via a mid-line neck incision and botharteries simultaneously occluded for 5 minutes with non-traumatic microaneurysm clips. After occlusion, the clips are removed to allow cerebralreperfusion and the neck incision closed with wound clips. All animalsare fasted overnight prior to the global ischemia surgery, a step thatfacilitates consistent ischemic injury. On Day 12, gerbils are exposedto a lethal dose of CO₂, and the brains frozen on dry ice and stored at−80° C.

[0116] The degree of neuronal protection is evaluated by in situhybridization analysis of neurogranin mRNA. Briefly, 20 μm coronalcryostat sections are collected on gelatin-coated slides, dried andstored at −80° C. At the time of processing, the desiccated slide boxesare warmed to room temperature, the slides postfixed in 4%paraformaldehyde, treated with acetic anhydride and then delipidated anddehydrated with chloroform and ethanol. Processed section-mounted slidesare then hybridized with 200 μl (6×10⁶ DPM/slide) of an antisense orsense (control) riboprobe for Neurogranin (³⁵S-UTP-labeled NG-241; bases99-340). in a 50% formamide hybridization mix and incubated overnight at55° C. in a humidified slide chamber without coverslipping. Thefollowing morning, the slides are collected in racks, immersed in 2×SSC(0.3 M NaCl, 0.03 M sodium citrate; pH 7.0)/ 10 mM DTT, treated withRNase A (20 μg/ml) and washed (2×30 min) at 67° C. in 0.1×SSC to removenonspecific label. After dehydration, the slides are opposed to BioMax(BMR-1; Kodak) X-ray film overnight.

[0117] The level of neurogranin hybridization signal is used toquantitatively assess the degree of neuronal loss in the CA1 regionafter injury and to evaluate the efficacy of 17β-estradiol andexperimental compounds. Neurogranin mRNA is selected for these studiesbecause it is highly expressed in the hippocampal neurons including CA1,but absent in glia and other cell types present in this brain region.Therefore, measurement of the amount of neurogranin mRNA presentrepresents surviving neurons. Relative optical density measurements ofneurogranin hybridization signal are obtained from film autoradiogramswith a computer based image analysis system (C-Imaging Inc., Pittsburgh,Pa.). The results from 6 sections (40 μm apart) per animal are averagedand statistically evaluated. Numerical values are reported as themean±SEM. One-way analysis of variance is used to test for differencesin the level of neurogranin mRNA and all statements of non-difference inthe results section imply that p>0.05.

[0118] Middle cerebral artery occlusion in mice. Neuroprotection can beevaluated according to the test procedures described by Dubal [see,Dubal, et al., Proceedings of the National Academy of Sciences of theUnited States of America 98: 1952-1957 (2001), Dubal, et al., Journal ofNeuroscience 19: 6385-6393 (1999)].

[0119] Ovulation Inhibition Standard Pharmacological Test Procedure

[0120] The test procedure is used to determine whether test compoundscan inhibit or change the timing of ovulation. It can also be used todetermine the number of oocytes ovulated [Lundeen, et al., J SteroidBiochem Mol Biol 78: 137-143 (2001)].

[0121] Based on the results obtained in the standard pharmacologicaltest procedures, the compounds of this invention are estrogen receptormodulators useful in the treatment or inhibition of conditions,disorders, or disease states that are at least partially mediated by anestrogen deficiency or excess, or which may be treated or inhibitedthrough the use of an estrogenic agent. The compounds of this inventionare particularly useful in treating a peri-menopausal, menopausal, orpostmenopausal patient in which the levels of endogenous estrogensproduced are greatly diminished. Menopause is generally defined as thelast natural menstrual period and is characterized by the cessation ofovarian function, leading to the substantial diminution of circulatingestrogen in the bloodstream. As used herein, menopause also includesconditions of decreased estrogen production that may be surgically,chemically, or be caused by a disease state which leads to prematurediminution or cessation of ovarian function.

[0122] Accordingly, the compounds of this invention are useful intreating or inhibiting osteoporosis and in the inhibition of bonedemineralization, which may result from an imbalance in a individual'sformation of new bone tissues and the resorption of older tissues,leading to a net loss of bone. Such bone depletion results in a range ofindividuals, particularly in post-menopausal women, women who haveundergone bilateral oophorectomy, those receiving or who have receivedextended corticosteroid therapies, those experiencing gonadaldysgenesis, and those suffering from Cushing's syndrome. Special needsfor bone, including teeth and oral bone, replacement can also beaddressed using these compounds in individuals with bone fractures,defective bone structures, and those receiving bone-related surgeriesand/or the implantation of prosthesis. In addition to those problemsdescribed above, these compounds can be used in treatment or inhibitionfor osteoarthritis, hypocalcemia, hypercalcemia, Paget's disease,osteomalacia, osteohalisteresis, multiple myeloma and other forms ofcancer having deleterious effects on bone tissues.

[0123] The compounds of this invention are also useful in inhibiting ortreating other effects of estrogen deprivation including, hot flushes,vaginal or vulvar atrophy, atrophic vaginitis, vaginal dryness,pruritus, dyspareunia, dysuria, frequent urination, urinaryincontinence, urinary tract infections. Other reproductive tract usesinclude the treatment or inhibition of dysfunctional uterine bleeding.

[0124] The compounds of this invention are also active in the brain andare therefore useful for inhibiting or treating Alzheimer's disease,cognitive decline, decreased libido, senile dementia, neurodegenerativedisorders, depression, anxiety, insomnia, schizophrenia, andinfertility. The compounds of this invention are also useful in treatingor inhibiting benign or malignant abnormal tissue growth including,glomerulosclerosis, prostatic hypertrophy, uterine leiomyomas, breastcancer, scleroderma, fibromatosis, endometriosis, endometrial cancer,polycystic ovary syndrome, endometrial polyps, benign breast disease,adenomyosis, ovarian cancer, melanoma, prostate cancer, cancers of thecolon, CNS cancers, such as glioma or astioblastomia.

[0125] The compounds of this invention are cardioprotective and areantioxidants, and are useful in lowering cholesterol, triglycerides,Lp(a), and LDL levels; inhibiting or treating hypercholesteremia,hyperlipidemia, cardiovascular disease, atherosclerosis, peripheralvascular disease, restenosis, and vasospasm, and inhibiting vascularwall damage from cellular events leading toward immune mediated vasculardamage. The compounds of this invention are also useful in treatingdisorders associated with inflammation or autoimmune diseases, includinginflammatory bowel disease (Crohn's disease, ulcerative colitis,indeterminate colitis), arthritis (rheumatoid arthritis,spondyloarthropathies, osteoarthritis), pleurisy, ischemia/reperfusioninjury (e.g. stroke, transplant rejection, myocardial infarction, etc.),asthma, giant cell arteritis, prostatitis, uveitis, psoriasis, multiplesclerosis, systemic lupus erythematosus and sepsis.

[0126] The compounds of this invention are also useful in treating orinhibiting ocular disorders including cataracts, uveitis, and maculardegeneration and in treating skin conditions such as aging, alopecia,and acne.

[0127] The compounds of this invention are also useful in treating orinhibiting metabolic disorders such as type-II diabetes, of lipidmetabolism, appetite (e.g. anorexia nervosa and bulimia).

[0128] Compounds in this invention are also useful in treating orinhibiting bleeding disorders such as hereditary hemorrhagictelangiectasia, dysfunctional uterine bleeding, and combatinghemorrhagic shock.

[0129] The compounds of this invention are useful in disease stateswhere amenorrhea is advantageous, such as leukemia, endometrialablations, chronic renal or hepatic disease or coagulation diseases ordisorders.

[0130] The compounds of this invention can be used as a contraceptiveagent, particularly when combined with a progestin.

[0131] When administered for the treatment or inhibition of a particulardisease state or disorder, it is understood that the effective dosagemay vary depending upon the particular compound utilized, the mode ofadministration, the condition, and severity thereof, of the conditionbeing treated, as well as the various physical factors related to theindividual being treated. Effective administration of the compounds ofthis invention may be given at an oral dose of from about 0.1 mg/day toabout 1,000 mg/day. Preferably, administration will be from about 10mg/day to about 600 mg/day, more preferably from about 50 mg/day toabout 600 mg/day, in a single dose or in two or more divided doses. Theprojected daily dosages are expected to vary with route ofadministration.

[0132] Such doses may be administered in any manner useful in directingthe active compounds herein to the recipient's bloodstream, includingorally, via implants, parentally (including intravenous,intraperitoneal, intraarticularly and subcutaneous injections),rectally, intranasally, topically, ocularly (via eye drops), vaginally,and transdermally.

[0133] Oral formulations containing the active compounds of thisinvention may comprise any conventionally used oral forms, includingtablets, capsules, buccal forms, troches, lozenges and oral liquids,suspensions or solutions. Capsules may contain mixtures of the activecompound(s) with inert fillers and/or diluents such as thepharmaceutically acceptable starches (e.g. corn, potato or tapiocastarch), sugars, artificial sweetening agents, powdered celluloses, suchas crystalline and microcrystalline celluloses, flours, gelatins, gums,etc. Useful tablet formulations may be made by conventional compression,wet granulation or dry granulation methods and utilize pharmaceuticallyacceptable diluents, binding agents, lubricants, disintegrants, surfacemodifying agents (including surfactants), suspending or stabilizingagents, including, but not limited to, magnesium stearate, stearic acid,talc, sodium lauryl sulfate, microcrystalline cellulose,carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginicacid, acacia gum, xanthan gum, sodium citrate, complex silicates,calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalciumphosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride,talc, dry starches and powdered sugar. Preferred surface modifyingagents include nonionic and anionic surface modifying agents.Representative examples of surface modifying agents include, but are notlimited to, poloxamer 188, benzalkonium chloride, calcium stearate,cetostearl alcohol, cetomacrogol emulsifying wax, sorbitan esters,colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesiumaluminum silicate, and triethanolamine. Oral formulations herein mayutilize standard delay or time release formulations to alter theabsorption of the active compound(s). The oral formulation may alsoconsist of administering the active ingredient in water or a fruitjuice, containing appropriate solubilizers or emulsifiers as needed.

[0134] In some cases it may be desirable to administer the compoundsdirectly to the airways in the form of an aerosol.

[0135] The compounds of this invention may also be administeredparenterally or intraperitoneally. Solutions or suspensions of theseactive compounds as a free base or pharmacologically acceptable salt canbe prepared in water suitably mixed with a surfactant such ashydroxy-propylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto inhibit the growth of microorganisms.

[0136] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0137] For the purposes of this disclosure, transdermal administrationsare understood to include all administrations across the surface of thebody and the inner linings of bodily passages including epithelial andmucosal tissues. Such administrations may be carried out using thepresent compounds, or pharmaceutically acceptable salts thereof, inlotions, creams, foams, patches, suspensions, solutions, andsuppositories (rectal and vaginal).

[0138] Transdermal administration may be accomplished through the use ofa transdermal patch containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin. The carrier may take any number of forms such as creams andointments, pastes, gels, and occlusive devices. The creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing the active ingredientmay also be suitable. A variety of occlusive devices may be used torelease the active ingredient into the blood stream such as asemi-permeable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

[0139] Suppository formulations may be made from traditional materials,including cocoa butter, with or without the addition of waxes to alterthe suppository's melting point, and glycerin. Water soluble suppositorybases, such as polyethylene glycols of various molecular weights, mayalso be used.

[0140] The preparation of representative examples of this invention isdescribed below.

[0141] Synthesis of Phenylboronic Acids

[0142] 4-Methoxy-2-trifluoromethylphenylboronic Acid

[0143] Step 1:

[0144] 4-Nitro-3-trifluoromethylanisole

[0145] A solution of 4-nitro-3-trifluoromethylphenol (27 g, 130 mmol) inacetone (250 mL) was treated with K₂CO₃ (18 g, 130 mmol) and methyliodide (16 mL, 260 mmol) and the mixture was heated at reflux undernitrogen. After 16 h, the cooled mixture was filtered to remove K₂CO₃and the filtrate was concentrated in vacuo to give the crude product (32g), which was taken up in benzene (250 mL). The organic solution waswashed with water and brine (125 mL each), dried (MgSO₄) andconcentrated in vacuo to give the title compound (27 g, 94%) as a yellowoil which was pure enough for further use.

[0146]¹H NMR (DMSO-d₆, 500 MHz) δ 3.95 (s, 3H, OCH ₃), 7.42-7.47(overlapping m, 2H, ArH), 8.19 (d, J=9 Hz, 1H, ArH).

[0147] Step 2:

[0148] 4-Amino-3-trifluoromethylanisole

[0149] A solution of 4-nitro-3-trifluoromethylanisole (26 g, 120 mmol)in ethyl acetate (250 mL) was hydrogenated over 10% palladium on carbon(5.3 g) at 50 psi for 1.5 h. At this time, the solution was filteredthrough Celite and washed with ethyl acetate (250 mL). The filtrate wasconcentrated in vacuo to give the title compound (22 g, 97%) as an amberoil which was pure enough for further use.

[0150]¹H NMR (DMSO-d₆, 500 MHz) δ 3.67 (s, 3H, OCH ₃), 5.07 (s, 2H, NH₂), 6.80 (d, J=9 Hz, 1H, ArH), 6.84 (d, J=3 Hz, 1H, ArH), 6.95 (m, 1H,ArH).

[0151] Step 3:

[0152] 4-Methoxy-2-trifluoromethylbenzenediazonium Tetrafluoroborate

[0153] A solution of 4-mino-3-trifluoromethylanisole (22 g, 110 mmol) inabsolute ethanol (200 mL) was treated with tetrafluoroboric acidsolution (54% in diethyl ether, 47 mL, 340 mmol) followed by technicalgrade t-butyl nitrite (90%, 18 mL, 140 mmol) at 0° C. under nitrogen.After 2.5 h at 0° C., the resulting precipitate was treated withice-cold diethyl ether (400 mL) and stirred for ca. 20 min. Vacuumfiltration gave the title compound (32 g, 95%) as white needles.

[0154]¹H NMR (DMSO-d₆, 300 MHz) δ 4.16 (s, 3H, OCH ₃), 7.80 (m, 1H,ArH), 8.01 (m, 1H, ArH), 8.99 (m, 1H, ArH).

[0155] Step 4:

[0156] 4-Bromo-3-trifluoromethylanisole

[0157] A solution of 4-methoxy-2-trifluoromethylbenzenediazoniumtetrafluoroborate (27 g, 93 mmol) in dimethyl sulfoxide (175 mL) wasadded dropwise during ca. 45 min to a vigorously stirred suspension ofCuBr₂ (70 g, 310 mmol) in dimethyl sulfoxide (400 mL) at 23° C. undernitrogen. After 16 h total, the dark mixture was diluted with water (1.2L) and extracted with benzene (3×300 mL). The combined extracts werewashed with brine (900 mL), dried (MgSO₄), and concentrated in vacuo togive a clear orange-brown liquid (27 g) which was flashed through a plugof silica gel (90 g) with hexane to remove baseline material.Concentration of the product-containing fractions gave the titlecompound (21 g, 85%) as a clear, almost colorless oil which was pureenough for further use.

[0158]¹H NMR (DMSO-d₆, 500 MHz) δ 3.82 (s, 3H, OCH ₃), 7.18 (m, 1H,ArH), 7.32 (d, J=3 Hz, 1H, ArH), 7.76 (d, J=9 Hz, 1H, ArH).

[0159] Step 5:

[0160] 4-Methoxy-2-trifluoromethylphenylboronic Acid

[0161] A solution of 4-bromo-3-trifluoromethylanisole (13 g, 50 mmol) indry THF (110 mL) was treated with butyllithium solution (2.5 M inhexane, 24 mL, 60 mmol) during ca. 5 min at −78° C. under nitrogen.After 15 min, the solution was treated with triisopropyl borate (23 mL,100 mmol) during ca. 5 min at −78° C. After a further 30 min, thecooling bath was removed and the reaction solution was warmed to 23° C.and left for 3.5 h. At this time, the resulting opaque mixture wasconcentrated in vacuo and the residue was treated with 1 N aqueoushydrogen chloride (110 mL, 110 mmol) and extracted with ethyl acetate(3×75 mL). The combined extracts were washed with water (2×100 mL) andbrine (100 mL), dried (Na₂SO₄), and concentrated in vacuo to give thecrude product as a tacky yellow solid (11 g) which was trituratedovernight with hexane (100 mL). Vacuum filtration gave the titlecompound (7.9 g, 72%) as a white solid which was pure enough for furtheruse.

[0162]¹H NMR (DMSO-d₆, 400 MHz) δ 3.80 (s, 3H, OCH ₃), 7.08-7.16(overlapping m, 2H, ArH), 8.12 (broad s, 2H, B(OH)₂).

[0163] 3-Chloro-4-methoxyphenylboronic Acid

[0164] Step 1:

[0165] 4-Bromo-2-chloroanisole

[0166] Starting from 4-bromo-2-chlorophenol (22 g, 110 mmol) in place of4-nitro-3-trifluoromethylphenol, the title compound (22 g, 93%, m.p.69-71° C.) was synthesized in essentially the same manner as describedabove for 4-nitro-3-trifluoromethylanisole in Step 1, Scheme 1.

[0167]¹H NMR (DMSO-d₆, 500 MHz) δ 3.84 (s, 3H, OCH ₃), 7.11 (d, J=9 Hz,1H, ArH), 7.49 (m, 1H, ArH), 7.65 (d, J=2 Hz, 1H, ArH). Analysis calc.for C₇H₆BrClO C, 37.96; H, 2.73 Found: C, 37.96; H, 2.66

[0168] Step 2:

[0169] 3-Chloro-4-methoxyphenylboronic Acid

[0170] Starting from 4-bromo-2-chloroanisole (5.0 g, 23 mmol) in placeof 4-bromo-3-trifluoromethylanisole, the title compound (2.6 g, 62%) wassynthesized in essentially the same manner as described above for4-methoxy-2-trifluoromethylphenylboronic acid in Step 2, Scheme 1.

[0171]¹H NMR (DMSO-d₆, 300 MHz) δ 3.82 (s, 3H, OCH ₃), 7.15 (m, 1H,ArH), 7.70 (m, 1H, ArH), 7.75 (m, 1H, ArH), 8.04 (broad s, 2H, B(OH)₂).

[0172] Synthesis of 2-(3-Bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane(Scheme 3)

[0173] Step 1:

[0174] 3-Bromo-2,5-diiodo-4-methylthiophene

[0175] A solution of 3-bromo-4-methylthiophene (51 g, 290 mmol) in 1:1chloroform-acetic acid (450 mL) was treated with N-iodosuccinimide (144g, 640 mmol, recrystallized from dioxane-carbon tetrachloride) at 23° C.and the mixture was stirred for 48 h. At this time, the resultingsolution was diluted with chloroform (4 L) and washed with water, 25%aqueous KOH and brine (1.5 L each), dried (K₂CO₃) and concentrated invacuo to give the crude product (106 g) as a dark oily solid which wastriturated with methanol (400 mL). Vacuum filtration gave the titlecompound (65 g, 52%, m.p. 81-82° C.) as a mustard yellow solid.

[0176]¹H NMR (CDCl₃, 400 MHz) δ 2.34 (s, ArCH ₃).

[0177] MS (APCI) m/z 428 (M⁻) Analysis calc. for C₅H₃BrI₂S C, 14.00; H,0.71 Found: C, 14.01; H, 0.57

[0178] Step 2:

[0179] 3-Bromo-5-iodo-4-methylthiophene-2-carbaldehyde

[0180] A suspension of 3-bromo-2,5-diiodo-4-methylthiophene (43 g, 100mmol) in diethyl ether (300 mL) was treated with an etheral solution of2.5 M butyllithium (40 mL, 100 mmol) during 5 min at −70 to −60° C.After 10 min, the mixture was treated with N,N-dimethylformamide (11 mL,140 mmol) and the cooling bath was removed. Upon warming to 23° C., themixture was quenched with 10% aqueous NH₄Cl (500 mL) and extracted withethyl acetate (3×500 mL). The combined extracts were washed with waterand brine (500 mL each), dried (MgSO₄) and concentrated in vacuo to givethe crude product (31 g) as a rust-colored solid which wasrecrystallized from hot ethyl acetate. Vacuum filtration gave the titlecompound (25 g, 76%, m.p. 152-53° C.) as rust-colored needles.

[0181]¹H NMR (CDCl₃, 400 MHz) δ 2.31 (s, 3H, ArCH ₃), 9.84 (s, 1H, CHO).MS (APCI) m/z 331 ([M+H]⁺) Analysis calc. for C₆H₄BrIOS C, 21.77; H,1.22 Found: C, 21.85; H, 1.08

[0182] Step 2:

[0183] 2-(3-Bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane

[0184] 3-Bromo-5-iodo-4-methylthiophene-2-carbaldehyde (27 g, 82 mmol),ethylene glycol (5.9 mL, 110 mmol) and p-toluenesulfonic acidmonohydrate (0.50 g, 2.6 mmol) were combined in benzene (200 mL) and themixture was heated at reflux under Dean-Stark conditions for 8 h. Atthis time, the cooled solution was washed with 2.5 N aqueous NaOH (200mL), water (4×200 mL) and brine (200 mL), dried (K₂CO₃) and concentratedin vacuo to give the title compound (29 g, 94%) as an orange oil.

[0185]¹H NMR (DMSO-d₆, 300 MHz) δ 2.16 (s, 3H, ArCH ₃), 3.90-4.04(overlapping m, 4H, (OCHH)₂), 5.99 (s,1H, CH).

[0186] MS (APCI) m/z 375 ([M+H]⁺) Analysis calc. for C₈H₈BrIO₂S C,25.62; H, 2.15 Found C, 25.43; H, 2.00

EXAMPLE 1

[0187]3-(3-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0188] Step 1:

[0189] 2-[3-Bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane

[0190] 2-(3-Bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane (12.4 g, 33.1mmol), 4-methoxyphenylboronic acid (5.5 g, 36 mmol), K₂CO₃ (15 g, 110mmol) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium [II](1.3 g, 1.6 mmol) were combined in 20% aqueous dimethoxyethane (130 mL)and heated at 75° C. After 3 h, the cooled mixture was filtered throughCelite and washed with ethyl acetate (350 mL). The filtrate was washedwith 1 N aqueous NaOH and brine (350 mL each), dried (K₂CO₃) andconcentrated in vacuo to give the crude product (13.1 g) as a dark solidwhich was pre-adsorbed on silica gel (40 g). Flash chromatography (600 gsilica, gradient 2.5%-5% ethyl acetate-hexane) gave the title compound(9.5 g, 81%, m.p. 103-04° C.) as a white crystalline solid.

[0191]¹H NMR (DMSO-d₆, 300 MHz) δ 2.18 (s, 3H, ArCH ₃), 3.79 (s, 3H, OCH₃), 3.93-4.08 (overlapping m, 4H, (OCHH)₂), 6.04 (s, 1H, CH), 7.30 (d,J=9 Hz, 2H, ArH), 7.39 (d, J=9 Hz, 2H, ArH).

[0192] MS (APCI) m/z 355 ([M+H]⁺) Analysis calc. for C₁₅H₁₅BrO₃S C,50.72; H, 4.26 Found: C, 50.41; H, 4.11

[0193] Step 2:

[0194]2-[3-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane

[0195] 2-[3-Bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane(7.0 g, 20 mmol), 3-methoxyphenylboronic acid (3.3 g, 22 mmol), K₂CO₃(9.1 g, 66 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium [II] (0.8 g, 1mmol) were combined in 20% aqueous dimethoxyethane (80 mL) and heated at75° C. After 8 h, the cooled mixture was filtered through Celite andwashed with ethyl acetate (200 mL). The filtrate was washed with 1 Naqueous NaOH and brine (200 mL each), dried (K₂CO₃) and concentrated invacuo to give the crude product (8.4 g) as a dark oil which waspre-adsorbed on silica gel (15 g). Flash chromatography (105 g silica,gradient 5%-10%-20% ethyl acetate-hexane) gave the title compound (7.2g, 95%) as a clear, colorless oil.

[0196]¹H NMR (DMSO-d₆, 300 MHz) δ 2.01 (s, 3H, ArCH ₃), 3.78 (s, 3H, OCH₃), 3.79 (s, 3H, OCH ₃), 3.85 (m, 2H, (OCHH)₂), 4.03 (m, 2H, (OCHH)₂),5.63 (s, 1H, CH), 6.88-6.91 (overlapping s, d, 2H, ArH), 6.97 (m, 1H,ArH), 7.03 (d, J=9 Hz, 2H, ArH), 7.35-7.43 (overlapping t, d, 3H, ArH).

[0197] MS (ESI) m/z 383 ([M+H]⁺) Analysis calc. for C₂₂H₂₂O₄S C, 69.09;H, 5.80 Found: C, 69.04; H, 5.65

[0198] Step 3:

[0199]3-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde

[0200]2-[3-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane(7.0 g, 18 mmol) and PPTS (1.4 g, 5.6 mmol) were combined in wet acetone(180 mL) and heated at reflux for 2 h. At this time, the cooled solutionwas concentrated in vacuo and the residue was taken up in ethyl acetate(200 mL). The organic phase was washed with saturated aqueous NaHCO₃ andbrine (200 mL each), dried (Na₂SO₄) and concentrated in vacuo to givethe title compound (5.9 g, 95%, m.p. 105-08° C.) as a yellow solid.

[0201]¹H NMR (DMSO-d₆, 300 MHz) δ 2.10 (s, 3H, ArCH ₃), 3.81 (s, 3H, OCH₃), 3.82 (s, 3H, OCH ₃), 7.02-7.10 (overlapping m, 5H, ArH), 7.44 (m,1H, ArH), 7.54 (d, J=9 Hz, 2H, ArH), 9.52 (s, 1H, CHO).

[0202] MS (ESI) m/z 339 ([M+H]⁺) Analysis calc. for C₂₀H₁₈O₃S C, 70.98;H, 5.36 Found: C, 70.83; H, 5.20

[0203] Step 4:

[0204]3-(3-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0205] A solution of3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde(3.9 g, 12 mmol) in dry methylene chloride (60 mL) was treated with amethylene chloride solution of 1.0 M boron tribromide (60 mL, 60 mmol)at 23° C. After 1.5 h, the reaction was quenched with saturated aqueousNaHCO₃ (200 mL) at 0° C. and extracted with ethyl acetate (2×100 mL).The combined extracts were washed with water and brine (150 mL each),dried (MgSO₄) and concentrated in vacuo to give a dark foam (3.7 g)which was pre-adsorbed on silica gel (15 g). Flash chromatography (75 gsilica, gradient 25%-50% ethyl acetate-hexane gave the title compound(3.3 g, 92%) as a yellow foam.

[0206]¹H NMR (DMSO-d₆, 300 MHz) δ 2.08 (s, 3H, ArCH), 6.81-6.91(overlapping m, 5H, ArH), 7.32 (t, J=8 Hz, 1H, ArH), 7.42 (d, J=9 Hz,2H, ArH), 9.50 (s, 1H, CHO), 9.72 (ex s, 1H, ArOH), 9.93 (ex s, 1H,ArOH).

[0207] MS (ESI) m/z 311 ([M+H]⁺) Analysis calc. for C₁₈H₁₄O₃S C, 69.66,H, 4.55 Found: C, 69.72; H, 4.49

EXAMPLE 2

[0208]3-(3-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0209] Starting from 2-(3-bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane(9.3 g, 25 mmol) and substituting 3-methoxyphenylboronic acid (4.1 g, 27mmol) in place of 4-methoxyphenylboronic acid (Example 1, Step 1) andsubstituting 4-methoxyphenylboronic acid in place of3-methoxyphenylboronic acid (Example 1, Step 2), the title compound(0.40 g, 85%, m.p. 208-10° C.) was synthesized in essentially the samemanner as described in Example 1.

[0210]¹H NMR (DMSO-d₆, 300 MHz) δ 2.11 (s, 3H, ArCH ₃), 6.84-7.00(overlapping m, 5H, ArH), 7.28-7.34 (overlapping m, 3H, ArH), 9.52 (s,1H, CHO), 9.81 (broad ex s, 2H, 2 ArOH).

[0211] MS (ESI) m/z 311 ([M+H]⁺) Analysis calc. for C₁₈H₁₄O₃S · 0.25EtOAc C, 68.66; H, 4.85 Found: C, 68.33; H, 4.83

EXAMPLE 3

[0212]3-(4-Hydroxy-2-methylphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde

[0213] Starting from2-[3-bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane (1.1 g,3.0 mmol, made in Example 1, Step 1) and substituting4-methoxy-2-methylphenylboronic acid (0.55 g, 3.3 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.56 g, 90%,m.p. 244-47° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0214]¹H NMR (DMSO-d₆, 500 MHz) δ 1.94 (s, 3H, ArCH ₃), 2.01 (s, 3H,ArCH ₃), 6.70 (d, J=8 Hz, 1H, ArH), 6.76 (s, 1H, ArH), 6.89 (d, J=8 Hz,2H, ArH), 7.04 (d, J=8 Hz, 1H, ArH), 7.44 (d, J=8 Hz, 2H, ArH), 9.31 (s,1H, CHO), 9.60 (ex s, 1H, ArOH), 9.93 (ex s, 1H, ArOH).

[0215] MS (ESI) m/z 323 ([M−H]⁻) Analysis calc. for C₁₉H₁₆O₃S · 0.05EtOAc C, 70.14; H, 5.03 Found: C, 69.82; H, 4.87

EXAMPLE 4

[0216]3-(5-Hydroxy-2-methylphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde

[0217] Starting from2-[3-bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane (1.1 g,3.0 mmol, made in Example 1, Step 1) and substituting5-methoxy-2-methylphenylboronic acid (0.55 g, 3.3 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.49 g, 89%,m.p. 204-07° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0218]¹H NMR (DMSO-d₆, 500 MHz) δ 1.94 (s, 3H, ArCH ₃), 1.95 (s, 3H,ArCH ₃), 6.62 (d, J=2 Hz, 1H, ArH), 6.79 (m, 1H, ArH), 6.89 (d, J=9 Hz,2H, ArH), 7.16 (d, J=8 Hz, 1H, ArH), 7.45 (d, J=9 Hz, 2H, ArH), 9.32 (s,1H, CHO), 9.46 (ex s, 1H, ArOH), 9.95 (ex s, 1H, ArOH). HRMS (ESI) calc.for C₁₉H₁₅O₃S ([M-H]⁻) 323.07474 Found: 323.07308

EXAMPLE 5

[0219]3-(3-Fluoro-4-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde

[0220] Starting from2-[3-bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane (1.1 g,3.0 mmol, made in Example 1, Step 1) and substituting3-fluoro-4-methoxyphenylboronic acid (0.56 g, 3.3 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.60 g, 97%,m.p. 223-26° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0221]¹H NMR (DMSO-d₆, 500 MHz) δ 2.09 (s, 3H, ArCH ₃), 6.90 (d, J=9 Hz,2H, ArH), 7.05-7.12 (overlapping m, 2H, ArH), 7.35 (m, 1H, ArH), 7.40(d, J=9 Hz, 2H, ArH), 9.51 (s, 1H, CHO), 9.94 (ex s,1H, ArOH), 10.3 (exs,1H, ArOH). HRMS (ESI) calc. for C₁₈H₁₂FO₃S ([M-H]⁻) 327.04966 Found:327.04793

EXAMPLE 6

[0222]3-(3-Formyl-4-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde

[0223] Starting from2-[3-bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane (1.5 g,4.2 mmol, made in Example 1, Step 1) and substituting3-formyl-4-methoxyphenylboronic acid (0.83 g, 4.6 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.26 g, 35%,m.p. 228-30° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0224]¹H NMR (DMSO-d₆, 500 MHz) δ 2.09 (s, 3H, ArCH ₃), 6.90 (d, J=9 Hz,2H, ArH), 7.14 (d, J=9 Hz, 1H, ArH), 7.42 (d, J=9 Hz, 2H, ArH), 7.65 (m,1H, ArH), 7.70 (d, J=9 Hz, 1H, ArH), 9.49 (s, 1H, CHO), 9.95 (ex s, 1H,ArOH), 10.3 (s, 1H, CHO), 11.1 (ex s, 1H, ArOH).

[0225] MS (ESI) m/z 337 ([M−H]⁻) Analysis calc. for C₁₉H₁₄O₄S C, 67.44;H, 4.17 Found: C, 67.09; H, 4.07

EXAMPLE 7

[0226]3-(3-Chloro-4-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0227] Starting from2-[3-bromo-5-(4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane (1.1 g,3.0 mmol, made in Example 1, Step 1) and substituting3-chloro-4-methoxyphenylboronic acid (0.67 g, 3.6 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.20 g, 36%,m.p. 234-36° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0228]¹H NMR (DMSO-d₆, 500 MHz) δ 2.08 (s, 3H, ArCH ₃), 6.89 (d, J=9 Hz,2H, ArH), 7.09 (d, J=8 Hz, 1H, ArH), 7.26 (m, 1H, ArH), 7.40 (d, J=9 Hz,2H, ArH), 7.49 (d, J=2 Hz, 1H, ArH), 9.50 (s, 1H, CHO), 9.92 (ex s, 1H,ArOH), 10.6 (ex s, 1H, ArOH). HRMS (ESI) calc. for C₁₈H₁₂ClO₃S ([M-H]⁻)343.02011 Found: 343.01921

EXAMPLE 8

[0229]5-(3-Fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde

[0230] Starting from 2-(3-bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane(2.3 g, 6.1 mmol) and 3-fluoro-4-methoxyphenylboronic acid (1.6 g, 9.4mmol) in place of 4-methoxyphenylboronic acid (Step 1), and substituting4-methoxyphenylboronic acid in place of 3-methoxyphenylboronic acid(Step 2), the title compound (0.42 g, 86%, m.p. 227-29° C.) wassynthesized in essentially the same manner as described in Example 1.

[0231]¹H NMR (DMSO-d₆, 500 MHz) δ 2.10 (s, 3H, ArCH ₃), 6.90 (d, J=9 Hz,2H, ArH), 7.07 (t, J=9 Hz, 1H, ArH), 7.23 (m, 1H, ArH), 7.28 (d, J=9 Hz,2H, ArH), 7.39 (m, 1H, ArH), 9.50 (s, 1H, CHO), 9.83 (ex s, 1H, ArOH),10.4 (ex s, 1H, ArOH).

[0232] MS (ESI) m/z 327 ([M−H]⁻) Analysis calc. for C₁₈H₁₃FO₃S C, 65.84;H, 3.99 Found: C, 65.67; H, 3.88

EXAMPLE 9

[0233]5-(3-Fluoro-4-hydroxyphenyl)-3-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehyde

[0234] Starting from2-[3-bromo-5-(3-fluoro-4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane(0.90 g, 2.4 mmol, made in Example 8, Step 1) and substituting4-methoxy-2-methylphenylboronic acid (0.44 g, 2.7 mmol) in place of4-methoxyphenylboronic acid (Step 2), the title compound (0.31 g, 76%,m.p. 227-30° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0235]¹H NMR (DMSO-d₆, 500 MHz) δ 1.95 (s, 3H, ArCH ₃), 2.01 (s, 3H,ArCH ₃), 6.70 (m, 1H, ArH), 6.76 (d, J=2 Hz, 1H, ArH), 7.03 (d, J=8 Hz,1H, ArH), 7.07 (t, J=9 Hz, 1H, ArH), 7.26 (m, 1H, ArH), 7.43 (m, 1H,ArH), 9.32 (s, 1H, CHO), 9.62 (ex s, 1H, ArOH), 10.4 (ex s, 1H, ArOH).HRMS (EI) calc. for C₁₉H₁₅FO₃S (M⁺) 342.0726 Found: 342.07259

EXAMPLE 10

[0236] 3-(3-Chloro-4-hydroxyphenyl)-5-(3-fluoro-4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0237] Starting from2-[3-bromo-5-(3-fluoro-4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane(0.89 g, 2.4 mmol, made in Example 8, Step 1) and substituting3-chloro-4-methoxyphenylboronic acid (0.53 g, 2.8 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.17 g, 33%,m.p. 252-54° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0238]¹H NMR (DMSO-d₆, 500 MHz) δ 2.09 (s, 3H, ArCH ₃), 7.06-7.10(overlapping m, 2H, ArH), 7.22-7.27 (overlapping m, 2H, ArH), 7.38 (m,1H, ArH), 7.49 (d, J=2 Hz, 1H, ArH), 9.51 (s, 1H, CHO), 10.4 (ex s, 1H,ArOH), 10.6 (ex s, 1H, ArOH).

[0239] MS (ESI) m/z 361 ([M−H]⁻) Analysis calc. for C₁₈H₁₂ClFO₃S C,59.59; H, 3.33 Found C, 59.35; H, 3.24

EXAMPLE 11

[0240]5-(4-Hydroxy-2-methylphenyl)-3-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde

[0241] Starting from 2-(3-bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane(2.5 g, 6.7 mmol) and 4-methoxy-2-methylphenylboronic acid (1.2 g, 7.2mmol) in place of 4-methoxyphenylboronic acid (Step 1), and substituting4-methoxyphenylboronic acid in place of 3-methoxyphenylboronic acid(Step 2), the title compound (0.50 g, 91%) was synthesized inessentially the same manner as described in Example 1.

[0242]¹H NMR (DMSO-d₆, 500 MHz) δ 1.83 (s, 3H, ArCH ₃), 2.11 (s, 3H,ArCH ₃), 6.68 (m, 1H, ArH), 6.75 (d, J=2 Hz, 1H, ArH), 6.89 (d, J=8 Hz,2H, ArH), 7.08 (d, J=8 Hz, 1H, ArH), 7.32 (d, J=8 Hz, 2H, ArH), 9.53 (s,1H, CHO), 9.70 (ex s, 1H, ArOH), 9.80 (ex s, 1H, ArOH).

[0243] MS (ESI) m/z 325 ([M+H]⁺) Analysis calc. for C₁₉H₁₆O₃S C, 70.35;H, 4.97 Found: C, 70.75; H, 5.36

EXAMPLE 12

[0244]3-(3-Fluoro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehyde

[0245] Starting from2-[3-bromo-5-(4-methoxy-2-methylphenyl)-4-methylthien-2-yl]-1,3-dioxolane(0.96 g, 2.6 mmol, made in Example 11, Step 1) and substituting3-fluoro-4-methoxyphenylboronic acid (0.57 g, 3.4 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.40 g, 67%)was synthesized in essentially the same manner as described in Example1, Steps 2-4.

[0246]¹H NMR (DMSO-d₆, 500 MHz) δ 1.84 (s, 3H, ArCH ₃), 2.11 (s, 3H,ArCH ₃), 6.68 (m, 1H, ArH), 6.75 (d, J=2 Hz, 1H, ArH), 7.04-7.08(overlapping m, 2H, ArH), 7.13 (m, 1H, ArH), 7.38 (m, 1H, ArH), 9.55 (s,1H, CHO), 9.70 (ex s, 1H, ArOH), 10.2 (ex s, 1H, ArOH).

[0247] MS (ESI) m/z 341 ([M−H]⁻) Analysis calc. for C₁₉H₁₅FO₃S · 0.22EtOAc C, 66.00; H, 4.67 Found: C, 65.63; H, 4.51

EXAMPLE 13

[0248]5-(3-Chloro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0249] Starting from 2-(3-bromo-5-iodo-4-methylthien-2-yl)-1,3-dioxolane(2.5 g, 6.7 mmol) and 3-chloro-4-methoxyphenylboronic acid (1.5 g, 8.0mmol) in place of 4-methoxyphenylboronic acid (Step 1), and substituting4-methoxyphenylboronic acid in place of 3-methoxyphenylboronic acid(Step 2), the title compound (0.41 g, 75%) was synthesized inessentially the same manner as described in Example 1.

[0250]¹H NMR (DMSO-d₆, 500 MHz) δ 2.09 (s, 3H, ArCH ₃), 6.90 (d, J=9 Hz,2H, ArH), 7.09 (d, J=8 Hz, 1H, ArH), 7.29 (d, J=9 Hz, 2H, ArH), 7.37 (m,1H, ArH), 7.54 (d, J=2 Hz, 1H, ArH), 9.50 (s, 1H, CHO), 9.83 (ex s, 1H,ArOH), 10.7 (ex s, 1H, ArOH).

[0251] MS (ESI) m/z 345 ([M+H]⁺) Analysis calc. for C₁₈H₁₃ClO₃S · 0.95CH₂Cl₂ C, 53.49; H, 3.53 Found: C, 53.53; H, 3.45

EXAMPLE 14

[0252]5-(3-Chloro-4-hydroxyphenyl)-3-(3-fluoro-4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0253] Starting from2-[3-bromo-5-(3-chloro-4-methoxyphenyl)-4-methylthien-2-yl]-1,3-dioxolane(1.0 g, 2.6 mmol, made in Example 13, Step 1) and substituting3-fluoro-4-methoxyphenylboronic acid (0.52 g, 3.1 mmol) in place of3-methoxyphenylboronic acid (Step 2), the title compound (0.26 g, 51%,m.p. 227-29° C.) was synthesized in essentially the same manner asdescribed in Example 1, Steps 2-4.

[0254]¹H NMR (DMSO-d₆, 500 MHz) δ 2.09 (s, 3H, ArCH ₃), 7.06-7.12(overlapping m, 3H total, ArH), 7.33-7.38 (overlapping m, 2H, ArH), 7.53(d, J=2 Hz, 1H, ArH), 9.52 (s, 1H, CHO), 10.3 (ex s, 1H, ArOH), 10.7 (exs, 1H, ArOH).

[0255] MS (ESI) m/z 363 ([M+H]⁺) Analysis calc. for C₁₈H₁₂ClFO₃S C,59.59; H, 3.33 Found: C, 59.60; H, 3.21

EXAMPLE 15

[0256] 3,5-Bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde

[0257] Starting from 3-bromo-5-iodo-4-methylthiophene-2-carbaldehyde(3.2 g, 9.7 mmol, Compound III) and 4-methoxyphenylboronic acid (3.2 g,21 mmol, 2.2 eq), the title compound (3.3 g, 94%, m.p. 223-25° C.) wassynthesized in essentially the same manner as described in Example 1,Steps 1 and 4.

[0258]¹H NMR (DMSO-d₆, 300 MHz) δ 2.09 (s, 3H, ArCH ₃), 6.89 (d, J=9 Hz,4H, ArH), 7.29 (d, J=9 Hz, 2H, ArH), 7.41 (d, J=9 Hz, 2H, ArH), 9.49 (s,1H, CHO), 9.81 (ex s, 1H, ArOH), 9.94 (ex s, 1H, ArOH).

[0259] MS (APCI) m/z 311 ([M+H]⁺) Analysis calc. for C₁₈H₁₄O₃S C, 69.66;H, 4.55 Found: C, 69.37; H, 4.51

EXAMPLE 16

[0260]3-(3-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime

[0261] Step 1:

[0262]3-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehydeOxime

[0263] A suspension of3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde(1.5 g, 4.4 mmol), hydroxylamine hydrochloride (0.40 g, 5.8 mmol) andpyridine (0.47 mL, 5.8 mmol) in ethanol (15 mL) was heated at reflux for1.5 h. The resulting solution was cooled to 23° C. and concentrated invacuo to give a viscous oil which was taken up in methylene chloride(100 mL). The organic solution was washed with water (3×50 mL) and brine(50 mL), dried (MgSO₄) and concentrated in vacuo to give a pale yellowfoam (1.4 g) which was pre-adsorbed on silica gel (3 g). Flashchromatography (30 g silica gradient 10%-20%-40% ethyl acetate-hexane)gave the title compound (1.3 g, 81%, m.p. 122-41° C.) as a pale yellowsolid (Mixture of E/Z isomers).

[0264]¹H NMR (DMSO-d₆, 500 MHz) δ 2.03 (s, 3H, ArCH ₃), 3.79 (s, 3H, OCH₃), 3.80 (s, 3H, OCH ₃), 6.86-6.88 (overlapping m, 2H, ArH), 7.00-7.06(overlapping m, 3H, ArH), 7.27 and 7.79 (s, 1H, CH═N), 7.39-7.47(overlapping m, 3H, ArH), 11.2 and 12.0 (ex s, 1H total, NOH).

[0265] MS (ESI) m/z 354 ([M+H]⁺) Analysis calc. for C₂₀H₁₉NO₃S C, 67.97;H, 5.42; N, 3.96 Found: C, 67.58; H, 5.07; N, 3.84

[0266] Step 2

[0267]3-(3-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeOxime

[0268] Starting from3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehydeoxime (1.2 g, 3.3 mmol) in place of3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde,the title compound (1.0 g, 93%) was synthesized in essentially the samemanner as described in Example 1, Step 4 (5:1 mixture of E/Z isomers).

[0269]¹H NMR (DMSO-d₆, 500 MHz) δ 1.98 and 2.01 (s, 3H total, ArCH ₃),6.66 (s, 1H, ArH), 6.70 (d, J=7 Hz, 1H, ArH), 6.81-6.86 (overlapping m,3H, ArH), 7.25-7.35 (overlapping m, s) and 7.79 (s, 4H, ArH, CH═N), 9.62(ex s, 1H, ArOH), 9.71 (ex s, 1H, ArOH), 11.4 and 11.9 (s, 1H total,NOH).

[0270] MS (ESI) m/z 326 ([M+H]⁺) Analysis calc. for C₁₈H₁₅NO₃S · C,64.32; H, 5.45; N, 3.54 0.80 EtOAc Found: C, 64.31; H, 5.42; N, 3.64

EXAMPLE 17

[0271]3-(3-Hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbonitrile

[0272] A mixture of3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime (0.76 g, 2.3 mmol) and pyridinium hydrochloride (2.7 g, 23 mmol)was heated at 195-205° C. in a 200 mL round bottom flask fitted with a250 mL bump trap. After 2 h, the reaction mixture was cooled to 23° C.and taken up in a minimum of ethyl acetate (100 mL). The organic phasewas washed with water (2×50 mL), saturated aqueous NaHCO₃ (50 mL) andbrine (50 mL), dried (Na₂SO₄) and concentrated in vacuo to give thecrude product as a brown solid (0.61 g) which was pre-adsorbed on silicagel (2 g). Flash chromatography (10 g silica gradient 20%-30%-40% ethylacetate-hexane) gave the title compound (0.37 g, 52%, m.p. 220-22° C.)as a pale orange solid after recrystallization from hot ethylacetate-hexane.

[0273]¹H NMR (DMSO-d₆, 500 MHz) δ 2.11 (s, 3H, ArCH ₃), 6.84-6.91(overlapping m, 5H, ArH), 7.33 (t, J=8 Hz, 1H, ArH), 7.39 (d, J=9 Hz,2H, ArH), 9.76 (ex s, 1H, ArOH), 9.94 (ex s, 1H, ArOH).

[0274] MS (ESI) m/z 308 ([M+H]⁺) Analysis calc. for C₁₈H₁₃NO₂S C, 70.34;H, 4.26; N, 4.56 Found: C, 70.10; H, 4.19; N, 4.43

EXAMPLE 18

[0275] 3,5-Bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde Oxime

[0276] Starting from3,5-bis(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde (2.0 g, 5.9mmol) in place of3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde,the title compound (0.91 g, 89%, m.p. 233-34° C.) was synthesized inessentially the same manner as described in Example 16 (Mixture of E/Zisomers).

[0277]¹H NMR (DMSO-d₆, 300 MHz) δ 1.98 and 2.00 (s, 3H total, ArCH ₃),6.84-6.87 (overlapping m, 4H, ArH), 7.09-7.12 (overlapping m, 2H, ArH),7.25 and 7.78 (s, 1H, CH═N), 7.30-7.34 (overlapping m, 2H, ArH), 9.65(ex s, 1H, ArOH), 9.71 and 9.74 (ex s, 1H total, ArOH), 11.1 and 11.9(s, 1H total, NOH). HRMS (Cl) calc. for C₁₈H₁₆NO₃S ([M + H]⁺) 326.0851Found: 326.0840

EXAMPLE 19

[0278] 3,5-Bis(4-hydroxyphenyl)-4-methylthiophene-2-carbonitrile

[0279] Starting from3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde oxime (0.70 g,2.2 mmol) in place of3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime, the title compound (0.33 g, 50%, m.p. 282-84° C.) was synthesizedin essentially the same manner as described in Example 17.

[0280]¹H NMR (DMSO-d₆, 300 MHz) δ 2.11 (s, 3H, ArCH ₃), 6.89-6.92(overlapping m, 4H, ArH), 7.31 (d, J=9 Hz, 2H, ArH), 7.38 (d, J=9 Hz,2H, ArH), 9.85 (ex s, 1H, ArOH), 9.92 (ex s, 1H, ArOH). Analysis calc.for C₁₈H₁₃NO₂S 0.10 EtOHC C, 70.07; H, 4.39; N, 4.49 Found: C, 69.91; H,4.33; N, 4.44

EXAMPLE 20

[0281] 3,5-Bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeO-methyloxime

[0282] Starting from3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde (0.86 g, 2.8mmol) in place of3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehydeand methoxylamine hydrochloride (0.25 g, 3.0 mmol) in place ofhydroxylamine hydrochloride, the title compound (0.59 g, 62%, m.p.219-25° C.) was synthesized in essentially the same manner as describedin Example 16, Step 1. Contains 6% E form as determined by NOE.

[0283]¹H NMR (DMSO-d₆, 500 MHz) δ 2.01 (s, 3H, ArCH ₃), 3.80 and 3.94(s, 3H, OCH ₃), 6.85-6.88 (overlapping m, 3H, ArH), 7.11 (d, J=8 Hz, 2H,ArH), 7.24 and 7.82 (s, 1H, CH═N), 7.33-7.35 (overlapping m, 3H, ArH),9.70 (ex s, 1H, ArOH), 9.76 (ex s, 1H, ArOH). HRMS (CI) calc. forC₁₉H₁₈NO₃S ([M + H]⁺) 340.1007 Found: 340.1004

EXAMPLE 21

[0284]3-(4-Hydroxyphenyl)-5-(3-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeOxime

[0285] Starting from3-(4-methoxyphenyl)-5-(3-methoxyphenyl)-4-methylthiophene-2-carbaldehyde(1.5 g, 4.4 mmol) in place of3-(3-methoxyphenyl)-5-(4-methoxyphenyl)-4-methylthiophene-2-carbaldehyde,the title compound (0.89 g, 82%) was synthesized in essentially the samemanner as described in Example 16 (7:1 mixture E/Z isomers).

[0286]¹H NMR (DMSO-d₆, 500 MHz) δ 1.98 and 2.04 (s, 3H total, ArCH ₃),6.78 (d, J=8 Hz, 1H, ArH), 6.87-6.94 (overlapping m, 4H, ArH), 7.12 (d,J=8 Hz, 2H, ArH), 7.24-7.27 and 7.80 (overlapping s, m, 2H, ArH, CH═N),9.63 (ex s, 1H, ArOH), 9.66 (ex s, 1H, ArOH), 11.2 and 11.9 (s, 1H,NOH).

[0287] MS (ESI) m/z 326 ([M+H]⁺) Analysis calc. for C₁₈H₁₅NO₃S 0.40EtOAc C, 65.28; H, 5.09; N, 3.88 Found: C, 65.29; H, 5.12; N, 3.92

EXAMPLE 22

[0288]3-(4-Hydroxyphenyl)-5-(3-hydroxyphenyl)-4-methylthiophene-2-carbonitrile

[0289] Starting from3-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime (0.60 g, 1.8 mmol) in place of3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime, the title compound (0.14 g, 25%, m.p. 240-41° C.) was synthesizedin essentially the same manner as described in Example 17.

[0290]¹H NMR (DMSO-d₆, 500 MHz) δ 2.14 (s, 3H, ArCH ₃), 6.86-6.88 (m,1H, ArH), 6.91-6.93 (overlapping m, 3H, ArH), 6.97 (m, 1H, ArH),7.30-7.34 (overlapping m, 3H, ArH), 9.81 (ex s, 1H, ArOH), 9.87 (ex s,1H total, ArOH).

[0291] MS (ESI) m/z 308 ([M+H]⁺) Analysis calc. for C₁₈H₁₃NO₂S C, 70.34;H, 4.26; N, 4.56 Found: C, 70.00; H, 4.14; N, 4.43

EXAMPLE 23

[0292]3-(3-Fluoro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehydeO-methyloxime

[0293] Starting from3-(3-fluoro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehyde(0.30 g, 0.88 mmol) in place of3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde, the titlecompound (0.22 g, 67%, m.p. 172-77° C.) was synthesized in essentiallythe same manner as described above in Example 20. Contains 8% E form asdetermined by NOE.

[0294]¹H NMR (DMSO-d₆, 500 MHz) δ 1.76 (s, 3H, ArCH ₃), 2.08 and 2.11(s, 3H, ArCH ₃), 3.80 and 3.92 (s, 3H, OCH ₃), 6.65 (m, 1H, ArH), 6.73(d, J=2 Hz, 1H, ArH), 6.95 (m, 1H, ArH), 7.03-7.07 (overlapping m, 2H,ArH), 7.16 (m, 1H, ArH), 7.31 (s, 1H, CH═N), 9.57 (ex s, 1H, ArOH), 10.1(ex s, 1H, ArOH).

[0295] MS (ESI) m/z 372 ([M+H]⁺) Analysis calc. for C₂₀H₁₈FNO₃S C,64.67; H, 4.88; N, 3.77 Found: C, 64.27; H, 5.01; N, 3.63

What is claimed is:
 1. A compound of formula I having the structure,

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 2. The compoundaccording to claim 1, wherein X is S.
 3. The compound according to claim2, wherein R² is phenyl optionally substituted with 1-4 Y groups and R³is hydrogen or alkyl of 1-6 carbon atoms.
 4. The compound according toclaim 3, wherein Z is —CHO, —CN, —CH═NOH, or —CH═NOR⁴.
 5. The compoundaccording to claim 1, which is a)5-(3-hydroxyphenyl)-3-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde;b)3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde;c)3-(4-hydroxy-2-methylphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde;d)-3-(5-hydroxy-2-methylphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde;e)3-(3-fluoro-4-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde;f)3-(3-formyl-4-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde;g)3-(3-chloro-4-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde;h)5-(3-fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde;i)5-(3-fluoro-4-hydroxyphenyl)-3-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehyde;j)3-(3-chloro-4-hydroxyphenyl)-5-(3-fluoro-4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde;k)5-(4-hydroxy-2-methylphenyl)-3-(4-hydroxyphenyl)-4-methyl-2-thiophenecarbaldehyde;l)3-(3-fluoro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehyde;m)5-(3-chloro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde;n)5-(3-chloro-4-hydroxyphenyl)-3-(3-fluoro-4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde;o) 3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde; p)3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime; q)3-(3-hydroxyphenyl)-5-(4-hydroxyphenyl)-4-methylthiophene-2-carbonitrile;r) 3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde oxime; s)3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbonitrile; t)3,5-bis(4-hydroxyphenyl)-4-methylthiophene-2-carbaldehyde O-methyloxime;u)3-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)-4-methylthiophene-2-carbaldehydeoxime; v)3-(4-hydroxyphenyl)-5-(3-hydroxyphenyl)-4-methylthiophene-2-carbonitrile;w)3-(3-fluoro-4-hydroxyphenyl)-5-(4-hydroxy-2-methylphenyl)-4-methylthiophene-2-carbaldehydeO-methyloxime or a pharmaceutically acceptable salt thereof.
 6. A methodof treating or inhibiting osteoporosis or inhibiting bonedemineralization in a mammal in need thereof, which comprises providingto said mammal an effective amount of a compound of formula I, havingthe structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 7. A method oftreating or inhibiting inflammatory bowel disease, Crohn's disease,ulcerative proctitis, or colitis in a mammal in need thereof, whichcomprises providing to said mammal an effective amount of a compound offormula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 8. A method oftreating or inhibiting prostatic hypertrophy, uterine leiomyomas, breastcancer, endometriosis, endometrial cancer, polycystic ovary syndrome,endometrial polyps, benign breast disease, adenomyosis, ovarian cancer,melanoma, prostrate cancer, colon cancer, glioma or astioblastomia in amammal in need thereof, which comprises providing to said mammal aneffective amount of a compound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 9. A method oflowering cholesterol, triglycerides, Lp(a), or LDL levels; inhibiting ortreating hypercholesteremia; hyperlipidemia; cardiovascular disease;atherosclerosis; peripheral vascular disease; restenosis, or vasospasm;or inhibiting vascular wall damage from cellular events leading towardimmune mediated vascular damage in a mammal in need thereof, whichcomprises providing to said mammal an effective amount of a compound offormula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 10. A method ofproviding cognition enhancement or neuroprotection; or treating orinhibiting senile dementias, Alzheimer's disease, cognitive decline,stroke, anxiety, or neurodegenerative disorders in a mammal in needthereof, which comprises providing to said mammal an effective amount ofa compound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, -CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 11. A method oftreating or inhibiting free radical induced disease states in a mammalin need thereof, which comprises providing to said mammal an effectiveamount of a compound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 12. A method oftreating or inhibiting vaginal or vulvar atrophy; atrophic vaginitis;vaginal dryness; pruritus; dyspareunia; dysuria; frequent urination;urinary incontinence; urinary tract infections in a mammal in needthereof, which comprises providing to said mammal an effective amount ofa compound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 13. A method oftreating or inhibiting vasomotor symptoms in a mammal in need thereof,which comprises providing to said mammal an effective amount of acompound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, -Co₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 14. A method ofinhibiting conception in a mammal in need thereof, which comprisesproviding to said mammal an effective amount of a compound of formula I,having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 15. A method oftreating or inhibiting arthritis in a mammal in need thereof, whichcomprises providing to said mammal an effective amount of a compound offormula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 16. The methodaccording to claim 15, wherein the arthritis is rheumatoid arthritis,osteoarthritis, or spondyloarthropathies.
 17. A method of treating orinhibiting joint swelling or erosion; or treating or inhibiting jointdamage secondary to arthroscopic or surgical procedures in a mammal inneed thereof, which comprises providing to said mammal an effectiveamount of a compound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 18. A method oftreating or inhibiting infertility in a mammal in need thereof, whichcomprises providing to said mammal an effective amount of a compound offormula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 19. A method oftreating or inhibiting ischemia, reperfusion injury, asthma, pleurisy,multiple sclerosis, systemic lupus erythematosis, uveitis, sepsis,hemmorhagic shock, or type II diabetes in a mammal in need thereof,which comprises providing to said mammal an effective amount of acompound of formula I, having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof.
 20. Apharmaceutical composition which comprises a compound of formula I,having the structure

wherein, R¹ is phenyl optionally substituted with 1-4 Y groups; R² isphenyl optionally substituted with 1-4 Y groups, alkyl of 1-6 carbonatoms, alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; R³ is hydrogen, phenyloptionally substituted with 1-4 Y groups, alkyl of 1-6 carbon atoms,alkoxy of 1-6 carbon atoms, alkoxycarbonyl of 2-7 carbon atoms,alkylthio of 1-6 carbon atoms, haloalkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, haloalkenyl of 2-7 carbonatoms, or haloalkynyl of 2-7 carbon atoms; X is O, —CH═CH—, or S; Y is—OH, —OR⁴, halogen, —CN, —CO₂H, —CO₂R⁴, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, perfluoroalkylof 1-6 carbon atoms, or —COR⁴; Z is —CHO, —CN, —CO₂H, —CO₂R⁴, —CONR⁴R⁵,—NO₂, —CH═NR⁴, —CH═NOH, or —CH═NOR⁴; R⁴ and R⁵ are each, independently,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, or cycloalkyl of 3-8 carbon atoms; with the proviso thatat least one of R² or R³ is phenyl or phenyl susbstituted with 1-4 Ygroups; or a pharmaceutically acceptable salt thereof, and apharmaceutical carrier.